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1
Arosio, Giulia, Geeta G. Sharma, Matteo Villa, Mario Mauri, Ilaria Crespiatico, Diletta Fontana, Chiara Manfroni, et al. "Synergistic Drug Combinations Prevent Resistance in ALK+ Anaplastic Large Cell Lymphoma." Cancers 13, no. 17 (September 1, 2021): 4422. http://dx.doi.org/10.3390/cancers13174422.
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Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma characterized by expression of the oncogenic NPM/ALK fusion protein. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. In these patients, the ALK inhibitor crizotinib achieves high response rates, however 30–40% of them develop further resistance to crizotinib monotherapy, indicating that new therapeutic approaches are needed in this population. We here investigated the efficacy of upfront rational drug combinations to prevent the rise of resistant ALCL, in vitro and in vivo. Different combinations of crizotinib with CHOP chemotherapy, decitabine and trametinib, or with second-generation ALK inhibitors, were investigated. We found that in most cases combined treatments completely suppressed the emergence of resistant cells and were more effective than single drugs in the long-term control of lymphoma cells expansion, by inducing deeper inhibition of oncogenic signaling and higher rates of apoptosis. Combinations showed strong synergism in different ALK-dependent cell lines and better tumor growth inhibition in mice. We propose that drug combinations that include an ALK inhibitor should be considered for first-line treatments in ALK+ ALCL.
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Amit, Amin Dipak, Praechompoo Pongtornpipat, and Jonathan H. Schatz. "Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells." Blood 122, no. 21 (November 15, 2013): 3842. http://dx.doi.org/10.1182/blood.v122.21.3842.3842.
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Abstract Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase. Disclosures: No relevant conflicts of interest to declare.
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Wass, Maxi, Timo Behlendorf, Ulrike Gläser, Jörn Rüssel, Fredericke Güntsch, Jordan Karin, and Hans-Joachim Schmoll. "Crizotinib in ALK-Positive Diffuse Large B-Cell Lymphoma: A Case Report." Blood 120, no. 21 (November 16, 2012): 4862. http://dx.doi.org/10.1182/blood.v120.21.4862.4862.
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Abstract Abstract 4862 Background: Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) displays a rare and distinct variant of DLBCL characterized by very aggressive clinical course with an estimated median survival of only 11.0 months. Crizotinib, an ALK inhibitor, has demonstrated impressive clinical efficacy in patients with alk-rearranged tumors such as NSCLC. The potential role of Crizotinib in ALK-positive DLBCL has not been established yet. Case report: A 27-year-old female patient with initial advanced-stage IV ALK-positive DLBCL was primary refractory to 6 cycles of standard chemotherapy with CHOP. She was subsequently treated with different salvage combination chemotherapy regimens including high-dose chemotherapy with autologous stem-cell transplantation. However, disease followed a very aggressive course and relapse occurred fast after each regimen. 6 weeks after transplantation patient presented with right cervical lymphadenopathy and multiple cutaneous lesions. CT scan revealed additional bilateral cervical, axillary, mediastinal and abdominal lymphadenopathy and involvement of lung and liver. LDH levels were exorbitant elevated (233 mmol/l). On an individual base, our patient received the ALK inhibitor Crizotinib 250 mg per os twice daily. No glucocorticoids or other drugs with antineoplastic activity were co-administered. Within 72 hours LDH levels were halved (89 mmol/l). On day 8 cervical palpable lymphadenopathy disappeared. Initial erythematous papular cutaneous lesion decreased from 4.0 × 2.5 cm to a residual hyperpigmented macule of 1.5 × 2.5 cm. On day 16 LDH levels were almost within normal limits (6 mmol/l). However, remission was not sustained and patient developed resistance to Crizotinib. By day 21 after beginning the treatment with Crizotinib LDH levels increased again and patient presented with a swollen left arm due to lymphatic obstruction, detected by CT scan, which showed a general progressive disease, causing death one month later. Conclusion: To our knowledge, this is the first case which reports sensitivity in ALK-positive DLBCL to ALK inhibition with Crizotinib. Despite remission duration was short our results indicate a potential role of ALK in the pathogenesis of ALK-positive DLBCL and suggest ALK inhibition as a potentially promising treatment modality in the therapy of this rare and fatal disease. Disclosures: Off Label Use: Crizotinib is an ALK inhibitor, indicated for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive. Recently impressive clinical efficacy of Crizotinib has been reported in two patients with ALK-positive anaplastic large cell lymphoma. Based on these data we initiated the treatment with Crizotinib in our patient, who showed very aggressive clinical behavior and fulminant early recurrence of a ALK-positive diffuse large B-cell lymphoma after salvage chemotherapy regimens on an individual base.
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Shi, Wenyu, and Jian-Yong Li. "GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL." Blood 134, Supplement_1 (November 13, 2019): 4069. http://dx.doi.org/10.1182/blood-2019-130063.
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Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control>GSK single drug 30 mg/kg>GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.
5
Andraos, Elissa, Joséphine Dignac, and Fabienne Meggetto. "NPM-ALK: A Driver of Lymphoma Pathogenesis and a Therapeutic Target." Cancers 13, no. 1 (January 5, 2021): 144. http://dx.doi.org/10.3390/cancers13010144.
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Initially discovered in anaplastic large cell lymphoma (ALCL), the ALK anaplastic lymphoma kinase is a tyrosine kinase which is affected in lymphomas by oncogenic translocations, mainly NPM-ALK. To date, chemotherapy remains a viable option in ALCL patients with ALK translocations as it leads to remission rates of approximately 80%. However, the remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. It is therefore crucial to identify new and better treatment options. Nowadays, different classes of ALK tyrosine kinase inhibitors (TKI) are available and used exclusively for EML4-ALK (+) lung cancers. In fact, the significant toxicities of most ALK inhibitors explain the delay in their use in ALCL patients, who are predominantly children. Moreover, some ALCL patients do not respond to Crizotinib, the first generation TKI, or develop an acquired resistance months following an initial response. Combination therapy with ALK inhibitors in ALCL is the current challenge.
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Galimov, A. N., E. E. Lepik, A. V. Kozlov, A. G. Gevorgian, I. V. Kazantsev, T. V. Yukhta, V. V. Baikov, et al. "The treatment of relapsed/refractory anaplastic large cell lymphoma expressing the anaplastic lymphoma kinase: a single-center experience." Pediatric Hematology/Oncology and Immunopathology 22, no. 1 (February 14, 2023): 22–31. http://dx.doi.org/10.24287/1726-1708-2023-22-1-22-31.
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Anaplastic large cell lymphoma (ALCL) expressing the anaplastic lymphoma kinase (ALK) (ALK+ ALCL) is a rare type of lymphoma which comprises 10-15% of all non-Hodgkin lymphomas in children and 2–3% in young adults. Relapsed/refractory disease occurs even more rarely (25–40% of all cases). There is as yet no standard treatment for relapsed/refractory ALK+ ALCL. Patients with ALK+ ALCL usually present at advanced stages of the disease with extranodal involvement (skin, soft tissues, bones, lungs, liver, spleen and bone marrow) and B symptoms. ALK-positive ALCL affects males more often than females. There are two morphological variants: the common type (65% of cases) and the non-common type which is associated with a poorer prognosis. ALK+ ALCLs are often associated with t(2;5) and t(1;2), resulting in the formation of the NPM-ALK and the TPM3-ALK fusion proteins, respectively. Data about the treatment of relapsed/refractory ALK+ ALCL are limited. Earlier, targeted therapies (brentuximab vedotin (BV), ALK inhibitors) and risk-adapted chemotherapy followed by hematopoietic stem cell transplantation (HSCT) for remission consolidation were shown to be highly effective. A total of 15 patients with relapsed/refractory ALK+ ALCL were treated at the R.M. Gorbacheva Research Institute starting from 2002. Fourteen (93%) patients had ALK-positive ALCL of common morphology and one (7%) patient had the non-common variant (histiocytic). The study was approved by the Independent Ethics Committee and the Scientific Council of the Pavlov University. The expression of CD3 on tumor cells was assessed (CD3 positive: n = 4 (27%), CD3 negative: n = 8 (53%), no data: n = 3 (20%). The median age at the diagnosis was 26 years (11 months– 37 years). The median follow-up from the diagnosis was 9 years (1–19 years). Nine (60%) patients were aged > 18 years and six (40%) patients were aged < 18 years. There were 10 (67%) males and 5 (33%) females. At onset, 2 (13%) patients were diagnosed with early-stage disease (stage II), while the others were diagnosed with advanced-stage disease: 2 (13%) patients had stage III disease and 11 (74%) had stage IV disease. Staging was performed according to the St. Jude staging system (in children) and the Ann Arbour staging classification (in adults). Thirteen (86%) patients had extranodal involvement. Four (27%) patients had refractory disease (progression within the first three months or the absence of complete remission after the first-line treatment) and the rest 11 (73%) patients had recurrent ALK-positive ALCL. Six patients developed early relapse (< 12 months after remission was achieved); 5 patients had late relapse (after > 12 months of remission); local (1 site) and systemic relapses were diagnosed in 7 and 4 patients, respectively. Our patients received from 2 to 7 lines of treatment (the median is 4). In the first line of therapy, the patients were treated according to NHL-BFM based regimens (n = 9; 60%), the CHOP (n = 5; 33%), and the HyperCVAD (n = 1; 7%) protocols. In the second line of therapy, 8 (53%) patients were treated according to NHL-BFM based regimens; 2 (13%) patients were treated with GDP; 1 (7%) patient received DHAP chemotherapy; 1 (7%) patient received a combination of methotrexate and vinblastine (MTX + V); 1 (7%) patient received bendamustine as a single agent. Two (13%) patients were treated with chemotherapy in combination with targeted drugs (GDP + BV, n = 1; NHL-BFM + crizotinib, n = 1). As a third or subsequent line of treatment, the patients received a variety of chemotherapy regimens (n = 5; 33%) and chemotherapy in combination with targeted drugs (n = 10; 67%). Five (33%) patients underwent ALK-inhibitor therapy (crizotinib (n = 4) and ceritinib (n = 1)). Seven (46%) patients were treated with BV (BV as a single agent (n = 4) and BV + chemotherapy (n = 3)). The median number of treatment lines before autologous HSCT (auto-HSCT) and allogeneic HSCT (allo-HSCT) was 2 (2–3) and 3 (3–4), respectively. Auto-HSCT was carried out in 11 (73%) cases. Nine (60%) patients underwent allo-HSCT (from a matched unrelated donor (n = 6), from a matched related donor (n = 2), and from a haploidentical donor (n = 1)). One patient received NK cells from a haploidentical donor as maintenance. In 5 (33%) cases, alloHSCT was carried out following auto-HSCT. The conditioning regimens (CR) used for auto-HSCT included BEAM (carmustine – 300 mg/m2, etoposide – 800 mg/m2, Cytosar – 1600 mg/m2, melphalan – 140 mg/m2) – in 5 (45%) patients; BeEAM (bendamustine – 320 mg/m2, etoposide – 800 mg/m2, Cytosar – 1600 mg/m2, melphalan – 140 mg/m2) – in 5 (45%) patients; and BuCy (Cyclophosphan – 100 mg/kg, busulfan – 14 mg/kg) – in 1 (10%) case. Seven (78%) patients undergoing allo-HSCT received the FluBenda conditioning regimen (fludarabine – 90–150 mg/m2, bendamustine – 390 mg/m2) and post-transplant Cyclophosphan and calcineurin inhibitors for the prevention of graft-versus-host disease (GVHD) (n = 7; 78%); one (11%) patient received the FluMel regimen (fludarabine – 150 mg/m2, melphalan – 140 mg/m2) and CsA/MTX (Cyclosporin А, methotrexate) for GVHD prevention; and in 1 case (11%) data on the RC and GVHD prophylaxis were missing. Overall response to the second line of treatment was achieved in 10 (67%) patients, with complete response observed in 7 (47%) cases, and partial response –in 3 (20%) cases. Five out of the 7 patients treated with BV during different lines of therapy managed to achieve complete response. Four out of the 5 patients who had undergone treatment with ALK inhibitors, demonstrated complete response. The 10-year overall survival (OS) rate of the study patients reached 90% (95% confidence interval (CI) 47–99). The 10-year progression-free survival (PFS) rate after the second line of treatment was 39% (95% CI 13–64). The 10-year OS and PFS rates after auto-HSCT were 100% and 35% (95% CI 8–64) respectively. The 5-year OS following allo-HSCT was 85% (95% CI 33–98), while PFS was 60% (95% CI 19–85). Four out of the 11 patients who had undergone auto-HSCT relapsed, and 2 patients progressed. Median time to relapse/progression was 8 (6–27) months. Three out of the 9 patients who had been treated with allo-HSCT ended up relapsing (median time: 8 (6–17) months). Two patients achieved repeated remission (in one case, it was the result of treatment with ceritinib, while in the other case it became possible after the resection of the lesion, radiotherapy and prescription of crizotinib), and 1 study patient died as a result of disease progression 17 months after allo-HSCT. The 6 patients who had achieved complete remission before undergoing allo-HSCT, are still in CR. Five out of the 9 patients developed grade I–II acute GVHD with skin involvement but did not show any signs of chronic GVHD. The observed complications of chemotherapy and auto-HSCT were standard and manageable and were not the focus of attention in this study. Taking into account the high probability of developing resistance to ALK inhibitors and the high risk of relapse after treatment with BV, targeted therapy should be used to prepare patients for HSCT. The use of ALK inhibitors and BV in our study led to repeated remission in 80% and 71% patients respectively. It was demonstrated that in the majority of cases even heavily pretreated patients with ALK+ ALCL can be cured (which is not the case with other non-Hodgkin lymphomas), especially if allogenic HSCT (allo-HSCT) is carried out. Still, we think that auto-HSCT can also be considered for remission consolidation since OS rates following auto-HSCT and allo-HSCT are comparable (100% and 85%). Moreover, auto-HSCT can also be a valid option in the absence of a fully matched donor, i.e. when only an alternative (haploidentical or partially matched) donor is available, since the use of haploidentical HSCT in ALCL patients has not been studied well enough yet. Further research on the matter is warranted. It was demonstrated that a non-myeloablative conditioning regimen before allo-HSCT (FluBenda) could be opted for in patients with relapsed/refractory ALK+ ALCL and that it was similarly effective as myeloablative RC when compared with a historical control group. Disease status before HSCT was proven to have a statistically significant influence on PFS (the best prognosis was associated with complete remission). At the same time, other factors did not impact the prognosis. This may be explained by the relatively small number of patients included in the study. Relapsed/refractory ALK+ ALCL is a disease with a relatively good prognosis even in heavily pretreated patients. Targeted therapy is a very important and effective step in preparation for HSCT. Allo-HSCT is more effective than auto-HSCT but the latter can also be considered a valid therapeutic option.
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Redaelli, Sara, Francesca Farina, Alessandra Stasia, Monica Ceccon, Luca Mologni, Cristina Messa, Luca Guerra, et al. "High Response Rates To Crizotinib In Advanced, Chemoresistant ALK+ Lymphoma Patients." Blood 122, no. 21 (November 15, 2013): 368. http://dx.doi.org/10.1182/blood.v122.21.368.368.
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Abstract In hematological disorders ALK expression is present in >50% of Anaplastic Large Cell Lymphomas (ALCL) as a result of a t(2;5)(p23;q35) translocation that causes the ALK gene on chromosome 2 to fuse with the NPM gene on chromosome 5. ALK + ALCL respond to cytotoxic drugs, but relapses occur and bear a poor prognosis(Stein, Foss et al. Blood 96 3681-95 2000; Ferreri, Govi et al. Crit Rev Oncol Hematol 83 293-302 2012). ALK-positive large B-cell lymphoma (ALK+ LBCL) is a rare lymphoma with a most frequent t(2;17)(p23;q23) translocation responsible for Clathrin-ALK fusion protein(Swerdlow, Campo et al. 2 2008). Crizotinib is the first ALK inhibitor which entered clinical practice: it is an orally bioavailable small-molecule inhibitor active on the ALK and MET receptor tyrosine kinases. While the activity of crizotinib in ALK+ lung cancer is documented (Kwak, Bang et al. N Engl J Med 363 1693-703 2010)no report on long term effects of crizotinib in ALK+ lymphomas exists; impressive short-term therapeutic activity was reported in two patients (Gambacorti-Passerini, Messa et al. N Engl J Med 364 775-6 2011), but no long-term data are available. In the present study, crizotinib was administered (250 mg BID) as monotherapy to 11 ALK+ lymphoma patients, diagnosed with ALK+ Non-Hodgkin lymphoma (NHL) by immunohistochemistry and FISH. Nine patients had a ALCL histology while the remaining 2 were DLBCL Patients had a refractory or relapsed disease after at least one prior chemotherapy regimen and measurable disease. All had involvement at multiple sites (nodal and extranodal) as well as B symptoms and an ECOG performance score of 1-4. Response to therapy was assessed according to RECIST criteria (Therasse, Arbuck et al. J Natl Cancer Inst 92 205-16 2000) The Overall Response Rate (ORR) was 10/11 (91%, 95% CI: 60-99%) and included 9 CR (82%, 95% CI: 51-96%) and 1 PR. Evidence of response by PET/CAT scan was present as early as 12 days. B symptoms disappeared promptly and LDH levels normalized within 30 days after the start of crizotinib. Disease status at the latest follow-up (June 2013) is as follows: 4 patients are in CR under continuous crizotinib treatment; they also test negative by RT-PCR for NPM/ALK (Mussolin, Damm-Welk et al. Leukemia 27 416-22 2012). Three patients (2 with LBCL and 1 with ALCL) died due to disease progression; 1 patient obtained CR, relapsed after 2 months of treatment and is now in CR on continued brentuximab treatment (month 29); 1 patient obtained CR on crizotinib and after 2 months stopped treatment, received an alloBMT and is still in CR; 2 patients treated for relapses post alloBMT obtained CR and are still in CR but they stopped crizotinib after 8-10 months. The two patients with ALK+ LBCL died within 3 months; in those with ALCL the CR rate was 9/9 (100%, 95% CI, 74-100%) with a median duration of 10 months (range 2-37). The 3 years PFS and OS rates are 62% (95% CI, 35-85%) and 73% (95% CI, 40-93%) respectively, with a plateau in the curve after the initial 6 months. In two relapsed patients the kinase domain of NPM-ALK could be amplified from peripheral blood samples obtained at the time of relapse (month 5 and 2). Deep sequencing of these products revealed the presence of different mutations: Q1064R at high prevalence (95%,) in patient (pt) #2 and I1171N (33%) plus M1328I (14%) in pt #6. All these mutations were not present in samples obtained before crizotinib treatment. I1171N was already discovered in an in-vitro screening (Ceccon, Mologni et al. Mol Cancer Res 11 122-32 2012): it commands an intermediate level of resistance to crizotinib (RI: 5.8) which however is cross resistant with other anti-ALK TKI such as AP26113 and NVP-TAE684. The other two mutations were not previously described: they present a RI to crizotinib of 2.4 (M1328I) and 8.5 (Q1064R). Since these residues do not form direct contacts with crizotinib, they probably interact with different structures within the catalytic domain such as the hydrophobic R-spine (I1171N) (Ceccon, Mologni et al. Mol Cancer Res 11 122-32 2012), the activation loop (M1328I), or yet unidentified regions (Q1064R). In conclusion, these positive results extend our initial observation on two patients (Gambacorti-Passerini, Messa et al. N Engl J Med 364 775-6 2011) and provide long-term follow up data. Crizotinib exerted a potent antitumor activity in advanced ALK+ lymphoma and produced durable responses in this population of heavily pre-treated patients, with a benign safety profile. Disclosures: Gambacorti-Passerini: Pfizer: Consultancy, Research Funding; BMS: Consultancy.
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Redaelli, Sara, Monica Ceccon, Alessandra Pirola, Marco Peronaci, Carlo Gambacorti-Passerini, and Luca Mologni. "Synergistic Activity of ALK and mTOR Inhibitors for the Treatment of NPM-ALK Positive Lymphoma." Blood 126, no. 23 (December 3, 2015): 3710. http://dx.doi.org/10.1182/blood.v126.23.3710.3710.
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Abstract ALK-positive Anaplastic Large Cell Lymphoma (ALCL) is a subset of Non-Hodgkin Lymphoma that positively took advantage of crizotinib development, a selective ALK inhibitor. Crizotinib is able to block ALK kinase activity which is fundamental for cancer cell survival. Constitutively active ALK kinase triggers the activation of several pro-proliferative and pro-survival downstream pathways, such as PI3K/AKT/mTOR. Despite the positive impact of targeted therapy on ALCL treatment, resistant clones tend to be selected during therapy. Strategies to overcome resistance include the design of more potent and selective drugs and the use of combined therapy approaches that allow for the simultaneous targeting of more than one node essential for cancer cells survival. Following this strategy, we decided to investigate the effects of combined ALK/mTOR inhibition. We first observed a synergistic effect of the combination of ALK inhibitors (crizotinib, alectinib or PF-3922) with an mTOR inhibitor (temsirolimus) in proliferation assays. Interestingly, the synergistic effect was observed only in ALK+ cell lines (Karpas 299, SUDH-L1 and SUPM2), while no synergism was observed in ALK- cell line (U937) as well as in normal lymphocytes. The Combination Index (CI) ranged from 0.16 to 0.45 indicating a synergistic/strong synergistic effect, accordingly to Chou classification. (Chou, 2006) (Table 1). The positive cooperation resulted in an increased inhibition of mTOR effectors p70S6K, 4EBP1 and eIF4B compared to single agent treatment as observed in immunoblot analysis performed on Karpas 299 treated for 4 hours. At the cell cycle level, the use of the drugs in combination induced a sharp block in G0/G1 phase, as observed by propidium iodide analysis performed on Karpas 299 at 48-72 and 96 hours. Long term exposure of ALK+ cells to either alectinib or temsirolimus, led to the establishment of resistant cell lines, while the exposure to the combination prevented the selection of resistant clones. Interestingly, the cell line resistant to alectinib showed a marked increase of ALK both at mRNA and protein level. In vivo, nude mice were injected with Karpas 299 cells. As the tumor masses reached 150mm3, mice were randomized and treated for 12 days with the combination of PF-3922 (0.5 mg/kg, administered per os twice a day) and temsirolimus (i.p., 1 mg/kg every other day) or with the single agents. The combined treatment induced a faster regression of the tumor masses compared to the single agents-treated mice. Moreover, responses were sustained for a longer period of time, upon treatment stop. (Figure 1) In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for patients affected by ALK+ ALCL. Table 1. combination indexes from proliferation experiments. Synergism levels calculated accordingly to Chou, Pharmacological reviews, 2006 Crizotinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:1 0.42 0.35 0.33 0.37 Synergism SUDHL1 3:1 0.49 0.45 0.42 0.45 Synergism SUPM2 1:1 0.59 0.33 0.29 0.40 Synergism NPM-ALK- U937 1:1 >10 >10 >10 >10 Antagonism HD Lymphocytes 1:1 >10 >10 >10 >10 Antagonism Alectinib - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:3 0.56 0.29 0.15 0.33 Synergism SUDHL1 1:3 0.53 0.45 0.38 0.45 Synergism SUPM2 1:10 0.65 0.18 0.06 0.3 Strong synergism NPM-ALK- U937 1:3 1.5 >10 >10 >10 Antagonism HD Lymphocytes 1:3 1.2 2.7 7.2 3.7 Antagonism PF06463922 - Temsirolimus Cell lines Ratio Combination Index (CI) Average CI Synergism level EC50 EC75 EC90 NPM-ALK+ Karpas 299 1:10 0.30 0.15 0.15 0.20 Strong synergism SUDHL1 1:30 0.57 0.41 0.29 0.42 Synergism SUPM2 1:3 0.30 0.12 0.06 0.16 Strong synergism NPM-ALK- U937 1:10 3.2 5.39 >10 >10 Antagonism HD Lymphocytes 1:3 1.4 8.8 >10 >10 Antagonism Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Figure 1. in vivo experiment for the evaluation of the combines treatment. Tumors volume measurements ±SEM are presented Disclosures No relevant conflicts of interest to declare.
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Atabay, Elif, Qi Wang, Ambrogio Chiara, Taek-Chin Cheong, Silvia Peola, Geeta G. Sharma, Luca Mologni, Carlo Gambacorti-Passerini, Claudia Voena, and Roberto Chiarle. "Identifying Novel Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Anaplastic Large Cell Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 5060. http://dx.doi.org/10.1182/blood-2019-132188.
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INTRODUCTION: Anaplastic large cell lymphomas (ALCL) frequently carry oncogenic fusion proteins as a consequence of chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. The fusion protein resulting from the translocation between dimerization domain of nucleophosmin (NPM) and intracellular tyrosine kinase domain of ALK activates several signaling pathways, promoting cell growth, transformation, migration, and survival of the cells. Chemotherapy has been used as a standard treatment approach for ALCL patients, yet about 30% of patients relapse. A more specific treatment method is based on targeting ALK tyrosine kinase using tyrosine kinase inhibitors (TKIs). Crizotinib is an ALK TKI that is approved for the treatment of ALK-rearranged lung cancer and has received Breakthrough Therapy designation for lymphoma because of its high activity in chemo refractory ALCL. However, as for lung cancer, also ALCL patient develop crizotinib resistance due to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown by-pass mechanisms of crizotinib resistance in ALCL. METHODS: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days, DNA isolation and next generation sequencing was performed on crizotinib resistant cells to identify candidate genes depleted by the GeCKO screening. Top candidates were selected for validation assays and further analyses. RESULTS: We identified two phosphatases, PTPN1 and PTPN2, in different ALCL cell lines as consistent top hits. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generate immediate resistance to crizotinib in ALCL cell lines. Analysis of downstream pathways showed that while loss of PTPN1 activates primarily the MAPK pathway, loss of PTPN2 promotes persistent STAT3 and MAPK activation in ALK inhibited cells. Remarkably, in PTPN1 knockout cells we observed hyperactivation of SHP2, an oncogenic phosphatase that positively regulates the RAS-MAPK pathway. On the other hand, over-expression of PTPN1 and PTPN2 partially inhibited SHP2 phosphorylation. A treatment that combined crizotinib and the recently developed SHP2 inhibitor completely blocked the sustained ERK phosphorylation and reverted the crizotinib resistance observed in PTPN1 and PTPN2 deficient lymphoma cells. CONCLUSIONS: GeCKO library screen identified PTPN1 and PTPN2 as specific genes that mediate crizotinib resistance in ALCL cell lines. Loss of PTPN1 and PTPN2 drives resistance by activating MAPK and/or JAK-STAT pathway. Combined inhibition of SHP2 is a potent therapeutic approach to overcome resistance to crizotinib in ALCL cells. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.
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Au, Trang H., Courtney C. Cavalieri, and David D. Stenehjem. "Ceritinib: A primer for pharmacists." Journal of Oncology Pharmacy Practice 23, no. 8 (October 13, 2016): 602–14. http://dx.doi.org/10.1177/1078155216672315.
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Clinical pharmacists are important contributors to the care of patients with cancer; it is therefore critical for oncology clinical pharmacists to stay current with new anticancer therapies. This review summarizes the epidemiology and pathogenesis of non-small cell lung cancer, including the most common genetic alterations, as well as the mechanism of action, clinical development, pharmacodynamics and pharmacokinetics of the anaplastic lymphoma kinase inhibitor ceritinib for the treatment of patients with anaplastic lymphoma kinase-positive non-small cell lung cancer. Targeted therapies based on the presence of specific mutations are an important development in the treatment of non-small cell lung cancer. However, acquired resistance to the first anaplastic lymphoma kinase-inhibitor approved by the U.S. Food and Drug Administration, crizotinib, is observed in almost half of patients treated with it. Ceritinib is an oral anaplastic lymphoma kinase-inhibitor that has demonstrated more potent antitumor activity than crizotinib in preclinical models. It was granted accelerated approval in 2014 to treat anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer patients who have progressed on or are intolerant to crizotinib. Ceritinib represents an important alternative second-line therapy for patients with metastatic non-small cell lung cancer who have traditionally limited treatment options.
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Karaca Atabay, Elif, Carmen Mecca, Qi Wang, Chiara Ambrogio, Ines Mota, Nina Prokoph, Giulia Mura, et al. "Tyrosine phosphatases regulate resistance to ALK inhibitors in ALK+ anaplastic large cell lymphoma." Blood 139, no. 5 (February 3, 2022): 717–31. http://dx.doi.org/10.1182/blood.2020008136.
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Abstract Anaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.
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Okawa, Sachi, Takuo Shibayama, Atsushi Shimonishi, Jun Nishimura, Taichi Ozeki, Kenji Takada, Hiroe Kayatani, et al. "Success of Crizotinib Combined with Whole-Brain Radiotherapy for Brain Metastases in a Patient with Anaplastic Lymphoma Kinase Rearrangement-Positive Non-Small-Cell Lung Cancer." Case Reports in Oncology 11, no. 3 (November 29, 2018): 777–83. http://dx.doi.org/10.1159/000492150.
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Although crizotinib shows marked antitumor activity in anaplastic lymphoma kinase (ALK) rearrangement-positive non-small-cell lung cancer (NSCLC) patients, all treated patients ultimately develop resistance to this drug. Isolated central nervous system failure without progression at extracranial sites is a common progression pattern in ALK rearrangement-positive NSCLC patients treated with crizotinib. Here, we report the success of crizotinib combined with whole-brain radiotherapy in an ALK rearrangement-positive NSCLC patient who developed leptomeningeal carcinomatosis and progression of multiple brain metastases. Additionally, we focused on the mechanism involved by examining the plasma and cerebrospinal fluid concentrations of crizotinib in the present case.
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Liang, Caixia, Ningning Zhang, Qiaoyun Tan, Shuxia Liu, Rongrong Luo, Yanrong Wang, Yuankai Shi, and Xiaohong Han. "CT-707 Overcomes Resistance of Crizotinib through Activating PDPK1- AKT1 Pathway by Targeting FAK." Current Cancer Drug Targets 19, no. 8 (September 6, 2019): 655–65. http://dx.doi.org/10.2174/1568009618666181031152140.
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Background: Crizotinib established the position of anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKI) in the treatment of non-small cell lung cancer (NSCLC) while the therapy- resistance hindered those patients from benefitting continuously from the treatment. CT-707 is an inhibitor of ALK/focal adhesion kinase (FAK) and IGFR-1. H2228CR (crizotinib resistance, CR) and H3122CR NSCLC cell lines were generated from the parental cell line H2228 (EML4-ALK, E6a/b:A20, variant 3) and H3122(EML4-ALK, E13:A20, variant 1), respectively. Methods: We investigated the antitumor effects CT-707 exerted against H3122CR in vitro /vivo. Results: Importantly, our study provided evidence that CT-707 overcomes resistance to crizotinib through activating PDPK1-AKT1 pathway by targeting FAK. Meanwhile, by using an in-vivo H3122CR xenograft model, we found CT-707 inhibited tumor growth significantly without obvious side effects. Conclusion: These findings indicate that CT-707 may be a promising therapeutic agent against crizotinib- resistance in NSCLC.
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Zdzalik, D., B. Dymek, P. Gunerka, P. Grygielewicz, K. Dzwonek, M. Lamparska-Przybysz, and M. Wieczorek. "1016 NPM-ALK I1171T Mutation Confers Resistance to Crizotinib in Anaplastic Large Cell Lymphoma." European Journal of Cancer 48 (July 2012): S245. http://dx.doi.org/10.1016/s0959-8049(12)71632-5.
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Shelikhova, L. N., V. V. Fominykh, D. S. Abramov, N. V. Myakova, M. A. Maschan, and A. A. Maschan. "Use of crizotinib for refractory ALK-positive lymphomas." Terapevticheskii arkhiv 89, no. 7 (July 15, 2017): 51–56. http://dx.doi.org/10.17116/terarkh201789751-56.
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Aim. To evaluate the safety and efficacy of crizotinib used in pediatric patients with relapsed or refractory ALK-positive anaplastic large-cell lymphoma (ALCL). Subjects and methods. The paper describes the experience with crizotinib used in 8 patients with refractory ALK-ALCL before and after allogeneic hematopoietic stem cell transplantation (HSCT). Results. All the 8 (100%) patients treated with crizotinib were recorded to have complete responses, including complete metabolic ones (tumor disappearance as evidenced by positron emission tomography (PET)/computed tomography. Conclusion. Low and manageable toxicity of crizotinib and complete PET-negative responses in patients with resistant ALK lymphomas favor the need to test the drug as first-line therapy, by possibly decreasing the intensification of chemotherapy.
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Shang, Chuquan, Bardes Hassan, Moinul Haque, Yuqi Song, Jing Li, Dongzhe Liu, Eva Lipke, Will Chen, Sylvie Giuriato, and Raymond Lai. "Crizotinib Resistance Mediated by Autophagy Is Higher in the Stem-Like Cell Subset in ALK-Positive Anaplastic Large Cell Lymphoma, and This Effect Is MYC-Dependent." Cancers 13, no. 2 (January 7, 2021): 181. http://dx.doi.org/10.3390/cancers13020181.
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Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.
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Shang, Chuquan, Bardes Hassan, Moinul Haque, Yuqi Song, Jing Li, Dongzhe Liu, Eva Lipke, Will Chen, Sylvie Giuriato, and Raymond Lai. "Crizotinib Resistance Mediated by Autophagy Is Higher in the Stem-Like Cell Subset in ALK-Positive Anaplastic Large Cell Lymphoma, and This Effect Is MYC-Dependent." Cancers 13, no. 2 (January 7, 2021): 181. http://dx.doi.org/10.3390/cancers13020181.
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Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.
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Huang, Ling, Xinmiao Jiang, Hanguo Guo, Liu Sichu, Xiaojuan Wei, Chen Feili, Zhanli Liang, and Wenyu Li. "TRAF1-ALK Fusion Predicts Poor Prognosis for ALK Positive Anaplastic Large Cell Lymphoma Patients with Chemotherapy and ALK Inhibitor." Blood 134, Supplement_1 (November 13, 2019): 5224. http://dx.doi.org/10.1182/blood-2019-126541.
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Background: Relapsed/refractory anaplastic lymphoma kinase (ALK) positive anaplastic large cell lymphomas (ALCLs) respond to ALK inhibitors, but resistance, which bear a poor prognosis. No biomarkers were found to predict long duration of response to ALK inhibitors. The ALK gene was first identified as the fusion partner of the nucleophosmin (NPM1) gene in the recurrent t(2;5)(p23;q35) found in a subset of ALCL. However, several distinct ALK fusions which result in highly different characteristics have also been described in lymphomas. Methods: We retrospectively reviewed seven relapsed/refractory ALK positive ALCLs who received ALK inhibitors (six with Crizotinib and one with Alectinib) at Guangdong Provincial People's Hospital from June 2007 through March 2019. We did next generation sequencing (NGS) with paraffin-embedded tissue for two patients who quickly developed resistance. Results: Of the seven patients, four were male and three were female, with a median age of 18 years (range 15-51) old. The median line of therapies was four (range 3-7) and that of ALK inhibitor usage was three (range 2-5). The overall response rate was 7 of 7 (100%) and the median overall survival of 21.2 months (8.5-86 months). Three patients obtained complete response (CR) on Crizotinib and then received autologous stem cell transplantation, and are still CR. One patient obtained CR, but died of serious infection five months after allogeneic stem cell transplantation. One patient is in CR under continuous crizotinib administration. One patient received Crizotinib obtained CR, but three months later got progression disease, the NGS showed TNF receptor-associated factor 1 gene (TRAF1) exon 6-ALK exon 20 fusion junction. The last patient was CR on alectinib, but quickly developed resistance, with a progression free survival of 1 month, the NGS indicated TRAF1 exon 7-ALK exon 20 fusion. Conclusions: ALK inhibitors improved survival of relapsed/refractory ALK positive ALCLs. TRAF1-ALK fusion may predict poor clinical outcome to chemotherapy and ALK inhibitors. This ALK fusion may reflect a trend to aggressive behaviour in lymphomas. Disclosures Li: Guangdong Province Hospital: Employment.
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Rolfo, Christian, Ignacio Gil-Bazo, and Solange Peters. "Adverse Event Management in Anaplastic Lymphoma Kinase-positive Non-small Cell Lung Cancer." European Oncology & Haematology 11, no. 2 (2015): 94. http://dx.doi.org/10.17925/eoh.2015.11.02.94.
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The development of oncogene-directed targeted therapies represents a new paradigm in the treatment of non-small cell lung cancer (NSCLC), offering improved outcomes compared with chemotherapy. Rearrangements of the anaplastic lymphoma kinase (ALK) gene are major oncogenic drivers in a subset of NSCLC patients. Since its launch in 2011, the ALK inhibitor crizotinib has become the standard of care inALK-positive NSCLC, but resistance inevitably develops. Ceritinib and alectinib have received regulatory approval: the former in Europe, US and elsewhere in the world, the latter in Japan. ALK inhibitors target multiple pathways, and may therefore be associated with a wide range of adverse events (AEs), including gastrointestinal AEs, hepatotoxicity and, in the case of crizotinib and ceritinib, cardiac effects. While the majority of these AEs are reversible, manageable and not severe, it is important that both physician and patients are aware of toxicities to ensure prompt treatment. This article discusses the management of AEs in patients receiving currently approved ALK inhibitors, including treatment, regular monitoring, drug discontinuation or dose reduction and physician/patient education. Proactive management of AEs enhances patient quality of life and optimises the therapeutic index of these agents.
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Shaw, Alice T., and Jeffrey A. Engelman. "ALKin Lung Cancer: Past, Present, and Future." Journal of Clinical Oncology 31, no. 8 (March 10, 2013): 1105–11. http://dx.doi.org/10.1200/jco.2012.44.5353.
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In 2007, scientists discovered that anaplastic lymphoma kinase (ALK) gene rearrangements are present in a small subset of non–small-cell lung cancers. ALK-positive cancers are highly sensitive to small-molecule ALK kinase inhibitors, such as crizotinib. Phase I and II studies of crizotinib in ALK-positive lung cancer demonstrated impressive activity and clinical benefit, leading to rapid US Food and Drug Administration approval in 2011. Although crizotinib induces remissions and extends the lives of patients, cures are not achieved as resistance to therapy develops. In this review, we will discuss the history of this field, current diagnostic and treatment practices, and future challenges and opportunities to advance outcomes for patients with ALK-positive lung cancers.
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Sica, Antonello, Caterina Sagnelli, Beniamino Casale, Gino Svanera, Massimiliano Creta, Armando Calogero, Renato Franco, Evangelista Sagnelli, and Andrea Ronchi. "How Fear of COVID-19 Can Affect Treatment Choices for Anaplastic Large Cell Lymphomas ALK+ Therapy: A Case Report." Healthcare 9, no. 2 (January 31, 2021): 135. http://dx.doi.org/10.3390/healthcare9020135.
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Background: The t (2; 5) chromosomal rearrangement of the ALK gene with nucleophosmin 1 gene (NPM1), resulting in an NPM1–ALK fusion, was first demonstrated in 1994 in anaplastic large cell lymphoma, (ALCL), a T-cell lymphoma responsive to cyclophosphamide, abriblastine, vincristine and prednisone in approximately 80% of cases; refractory cases usually respond favorably to brentuximab vedotin. These treatments are regarded as a bridge to allogeneic hematopoietic stem cell transplantation (allo-SCT). Nowadays, transplant procedures and the monitoring of chemotherapy patients proceed very slowly because the SARS-CoV-2 pandemic has heavily clogged the hospitals in all countries. Results: A 40-year-old Caucasian woman was first seen at our clinical center in June 2020. She had ALCL ALK+, a history of failure to two previous therapeutic lines and was in complete remission after 12 courses of brentuximab, still pending allo-SCT after two failed donor selections. Facing a new therapeutic failure, we requested and obtained authorization from the Italian drug regulatory agency to administer 250 mg of crizotinib twice a day, a drug incomprehensibly not registered for ALCL ALK +. Conclusions: The response to crizotinib was optimal since no adverse event occurred, and CT-PET scans persisted negative; this drug has proved to be a valid bridge to allo-SCT.
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Mossé, Yael P., Stephan D. Voss, Megan S. Lim, Delphine Rolland, Charles G. Minard, Elizabeth Fox, Peter Adamson, Keith Wilner, Susan M. Blaney, and Brenda J. Weigel. "Targeting ALK With Crizotinib in Pediatric Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic Tumor: A Children’s Oncology Group Study." Journal of Clinical Oncology 35, no. 28 (October 1, 2017): 3215–21. http://dx.doi.org/10.1200/jco.2017.73.4830.
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Purpose Fusions involving the ALK gene are the predominant genetic lesion underlying pediatric anaplastic large cell lymphomas (ALCL) and inflammatory myofibroblastic tumors (IMTs). We assessed the activity of the ALK inhibitor crizotinib in patients who had no known curative treatment options at diagnosis or with relapsed/recurrent disease. Methods In this study, 26 patients with relapsed/refractory ALK-positive ALCL and 14 patients with metastatic or inoperable ALK-positive IMT received crizotinib orally twice daily. Study objectives were measurement of efficacy and safety. Correlative studies evaluated the serial detection of NPM-ALK fusion transcripts in patients with ALCL. Results The overall response rates for patients with ALCL treated at doses of 165 (ALCL165) and 280 (ALCL280) mg/m2 were 83% and 90%, respectively. The overall response rate for patients with IMT (treated at 100, 165, and 280 mg/m2/dose) was 86%. A complete response was observed in 83% (five of six) of ALCL165, 80% (16 of 20) of ALCL280, and 36% (five of 14) of patients with IMT. Partial response rates were 0% (none of six), 10% (two of 20), and 50% (seven of 14), respectively. The median duration of therapy was 2.79, 0.4, and 1.63 years, respectively, with 12 patients ceasing protocol therapy to proceed to transplantation. The most common drug-related adverse event was decrease in neutrophil count in 33% and 70% of the ALCL165 and ALCL280 groups, respectively, and in 43% of patients with IMT. Levels of NPM-ALK decreased during therapy in most patients with ALCL. Conclusion The robust and sustained clinical responses to crizotinib therapy in patients with relapsed ALCL and metastatic or unresectable IMT highlight the importance of the ALK pathway in these diseases.
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Dasari, Surendra, Kenneth L. Johnson, Carrie J. H. Hepplemann, Ariel J. Caride, Jason D. Theis, H. Robert Bergen, Ahmet Dogan, and Andrew L. Feldman. "Deep Proteomic Profiling Predicts Differential Chemosensitivity In Anaplastic Large Cell Lymphoma Cell Lines." Blood 122, no. 21 (November 15, 2013): 1670. http://dx.doi.org/10.1182/blood.v122.21.1670.1670.
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Abstract Background The lymphoma proteome is the phenotypic representation of the underlying genetic and epigenetic makeup of each individual patient's tumor. The proteome is rich in druggable targets and offers a unique opportunity for the hematologist to personalize therapy. We developed a deep proteomic profiling method using ion exchange fractionation and tandem mass spectrometry. In this pilot study, we applied this method to detect differentially expressed proteins in anaplastic large cell lymphoma (ALCL) cell lines with previously known (ALK-positive) and unknown (ALK-negative) therapeutic targets. We then asked whether integrative informatic analysis of these data could be used to predict drug sensitivity in each of the cell lines. Methods To examine reproducibility of our method, proteins were extracted independently from 4 pellets each of FE-PD (ALK-negative) and Karpas 299 (ALK-positive) ALCL cells, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Resulting peptides were separated into 6 fractions using strong anion exchange (SAX) chromatography. Peptides in each fraction were analyzed via shotgun proteomics on a QExactive mass spectrometer. Peptide mass spectra (MS/MS) were matched against a RefSeq human protein sequence database using MyriMatch software. Reversed sequences were added to the database to measure identification false discovery rates (FDRs). IDPicker filtered the peptide identifications at 2% FDR. Proteins with at least two unique peptide identifications and five MS/MS matches were considered to be present in the sample. Filtered protein identifications and corresponding spectral counts were used as input to QuasiTel software, which was configured to use proteins with at least one spectrum per biological replicate. Proteins that were significantly differentially expressed (quasi p-value < 0.05) with an absolute log2 fold-change of at least 0.5 fold were loaded into the Ingenuity Pathway Analysis (IPA) software, and a master list of drugs and corresponding gene targets was assembled using PharmGKB database and the Drug-Gene Interaction Database (DGID). The resulting drug-gene target list was merged with the differentially expressed protein identifications. Candidate targets were validated by Western blot and candidate drugs were assessed in viability assays. Results The SAX-LC-MS/MS method identified 10,111 proteins from all replicate analyses of FE-PD and Karpas 299 samples, and 93% of the identified proteome was detected in all 4 replicate analyses. The detected proteome was well represented by key transcription factors, phophatases, kinases, translation regulators and transmembrane regulators. There were 1369 proteins differentially expressed between the 2 cell lines, 709 up regulated in Karpas 299 cell line and 673 up regulated in FE-PD. Differentially expressed proteins also showed consistent expression across the biological replicates (Figure 1). Our integrated approach to identify candidate targets and drugs “rediscovered” ALK in Karpas299 and unexpectedly identified relative overexpression of the IL2-IL2RA-STAT5A-STAT5B network in ALK-negative FE-PD cells. Western blot confirmed these findings. As expected, 50-100 nM crizotinib (ALK inhibitor) decreased Karpas 299 viability (p =0.016) but had no effect on FE-PD (Figure 2). In contrast, 50-100 nM of the experimental STAT5 inhibitor 573108 (EMD Millipore) decreased FE-PD viability (p=0.002) but had no effect on Karpas 299 (Figure 2). Conclusion The lymphoma proteome is complex with 10,000 proteins and contains druggable targets that can be reproducibly identified using SAX-LC-MS/MS. These targets vary across samples and integrated informatic analysis can predict target-drug combinations that have efficacy in an experimental model. These data suggest that SAX-LC-MS/MS could be used to personalize treatment regimens in lymphoma patients. Figure 1: Spectral counts of 1369 differentially expressed proteins between Karpas299 and FE-PD cell lines were normalized and plotted in a heat map. Red color indicates down regulation and yellow color indicates up regulation. Disclosures: No relevant conflicts of interest to declare.
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Wurster, Kathrin, Mariantonia Costanza, Stephan Kreher, Selina Glaser, Björn Lamprecht, Nikolai Schleussner, Ioannis Anagnostopoulos, et al. "Aberrant Expression of and Cell Death Induction by Engagement of the MHC-II Chaperone CD74 in Anaplastic Large Cell Lymphoma (ALCL)." Cancers 13, no. 19 (October 7, 2021): 5012. http://dx.doi.org/10.3390/cancers13195012.
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In 50–60% of cases, systemic anaplastic large cell lymphoma (ALCL) is characterized by the t(2;5)(p23;q35) or one of its variants, considered to be causative for anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL. Key pathogenic events in ALK-negative (ALK−) ALCL are less well defined. We have previously shown that deregulation of oncogenic genes surrounding the chromosomal breakpoints on 2p and 5q is a unifying feature of both ALK+ and ALK− ALCL and predisposes for occurrence of t(2;5). Here, we report that the invariant chain of the MHC-II complex CD74 or li, which is encoded on 5q32, can act as signaling molecule, and whose expression in lymphoid cells is usually restricted to B cells, is aberrantly expressed in T cell-derived ALCL. Accordingly, ALCL shows an altered DNA methylation pattern of the CD74 locus compared to benign T cells. Functionally, CD74 ligation induces cell death of ALCL cells. Furthermore, CD74 engagement enhances the cytotoxic effects of conventional chemotherapeutics in ALCL cell lines, as well as the action of the ALK-inhibitor crizotinib in ALK+ ALCL or of CD95 death-receptor signaling in ALK− ALCL. Additionally, a subset of ALCL cases expresses the proto-oncogene MET, which can form signaling complexes together with CD74. Finally, we demonstrate that the CD74-targeting antibody-drug conjugate STRO-001 efficiently and specifically kills CD74-positive ALCL cell lines in vitro. Taken together, these findings enabled us to demonstrate aberrant CD74-expression in ALCL cells, which might serve as tool for the development of new treatment strategies for this lymphoma entity.
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Hare, Lucy, G. A. Amos Burke, and Suzanne D. Turner. "Resistance to Targeted Agents Used to Treat Paediatric ALK-Positive ALCL." Cancers 13, no. 23 (November 29, 2021): 6003. http://dx.doi.org/10.3390/cancers13236003.
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Non-Hodgkin lymphoma (NHL) is the third most common malignancy diagnosed in children. The vast majority of paediatric NHL are either Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), anaplastic large cell lymphoma (ALCL), or lymphoblastic lymphoma (LL). Multi-agent chemotherapy is used to treat all of these types of NHL, and survival is over 90% but the chemotherapy regimens are intensive, and outcomes are generally poor if relapse occurs. Therefore, targeted therapies are of interest as potential solutions to these problems. However, the major problem with all targeted agents is the development of resistance. Mechanisms of resistance are not well understood, but increased knowledge will facilitate optimal management strategies through improving our understanding of when to select each targeted agent, and when a combinatorial approach may be helpful. This review summarises currently available knowledge regarding resistance to targeted therapies used in paediatric anaplastic lymphoma kinase (ALK)-positive ALCL. Specifically, we outline where gaps in knowledge exist, and further investigation is required in order to find a solution to the clinical problem of drug resistance in ALCL.
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Davare, Monika A., Nadeem A. Vellore, Jacob P. Wagner, Christopher A. Eide, James R. Goodman, Alexander Drilon, Michael W. Deininger, Thomas O’Hare, and Brian J. Druker. "Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors." Proceedings of the National Academy of Sciences 112, no. 39 (September 8, 2015): E5381—E5390. http://dx.doi.org/10.1073/pnas.1515281112.
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Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.
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Sharma, Geeta, Ines Mota, Luca Mologni, Enrico Patrucco, Carlo Gambacorti-Passerini, and Roberto Chiarle. "Tumor Resistance against ALK Targeted Therapy-Where It Comes From and Where It Goes." Cancers 10, no. 3 (February 28, 2018): 62. http://dx.doi.org/10.3390/cancers10030062.
Full textAbstract:
Anaplastic lymphoma kinase (ALK) is a validated molecular target in several ALK-rearranged malignancies, particularly in non-small-cell lung cancer (NSCLC), which has generated considerable interest and effort in developing ALK tyrosine kinase inhibitors (TKI). Crizotinib was the first ALK inhibitor to receive FDA approval for ALK-positive NSCLC patients treatment. However, the clinical benefit observed in targeting ALK in NSCLC is almost universally limited by the emergence of drug resistance with a median of occurrence of approximately 10 months after the initiation of therapy. Thus, to overcome crizotinib resistance, second/third-generation ALK inhibitors have been developed and received, or are close to receiving, FDA approval. However, even when treated with these new inhibitors tumors became resistant, both in vitro and in clinical settings. The elucidation of the diverse mechanisms through which resistance to ALK TKI emerges, has informed the design of novel therapeutic strategies to improve patients disease outcome. This review summarizes the currently available knowledge regarding ALK physiologic function/structure and neoplastic transforming role, as well as an update on ALK inhibitors and resistance mechanisms along with possible therapeutic strategies that may overcome the development of resistance.
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Doebele, Robert Charles, Dara L. Aisner, Anh T. Le, Eamon M. Berge, Amanda B. Pilling, Tatiana G. Kutateladze, Andrew James Weickhardt, Wilbur A. Franklin, Marileila Varella-Garcia, and D. Ross Camidge. "Analysis of resistance mechanisms to ALK kinase inhibitors in ALK+ NSCLC patients." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 7504. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.7504.
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7504 Background: Patients with anaplastic lymphoma kinase (ALK) gene fusions derive significant clinical benefit from crizotinib, an ALK inhibitor; moreover, next generation ALK kinase inhibitors are in development. Unfortunately, drug resistance develops after initial benefit (acquired) or occurs in patients who never derive a benefit (intrinsic). This study aimed to define molecular mechanisms of resistance to ALK kinase inhibitors in ALK+ non-small cell lung cancer (NSCLC) patients. Methods: 30 ALK+ crizotinib-treated NSCLC patients experienced radiologic disease progression, of whom 7 progressed only in the CNS. Of the 23 patients with extra-CNS progression, a biopsy was attempted on 19. One of the patients without tissue post-crizotinib then proceeded to a second generation ALK inhibitor and tissue was obtained post progression on this drug. We performed molecular analysis and initiated cell lines from tumor tissue for these 19 patients. Results: 15 patients had material evaluable for molecular analysis. Six patients (40%) developed secondary mutations in the kinase domain of ALK. Two novel mutations were identified in samples from ALK+ NSCLC patients, F1174C and D1203N. Two patients each demonstrated G1269A or L1196M mutations. Two patients each, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). Four patients demonstrated the presence of another oncogenic driver (1 with EGFR mutation; 3 with KRAS mutation) with or without a persistent ALK gene rearrangement. One patient lacked an ALK gene fusion on progression biopsy, but had no identifiable alternate oncogene alteration. 3 patients retained ALK positivity with no identifiable resistance mechanism. Data on additional patients and an in vitro model of copy number gain will be presented. Conclusions: ALK kinase inhibitor resistance in ALK+ NSCLC occurs through a diverse array of kinase domain mutations, ALK gene fusion CNG, and emergence of second (same cell) or separate (different cell) oncogenic drivers. In order to overcome resistance it will be important to differentiate patients that preserve ALK dominance (secondary mutations and CNG) versus those that have diminished ALK dominance (separate or second oncogenic drivers).
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Gristina, Valerio, Maria La Mantia, Federica Iacono, Antonio Galvano, Antonio Russo, and Viviana Bazan. "The Emerging Therapeutic Landscape of ALK Inhibitors in Non-Small Cell Lung Cancer." Pharmaceuticals 13, no. 12 (December 18, 2020): 474. http://dx.doi.org/10.3390/ph13120474.
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The treatment of metastatic non-small cell lung cancer (NSCLC) has undergone a paradigm shift over the last decade. Better molecular characterization of the disease has led to the rapid improvement of personalized medicine and the prompt delivery of targeted therapies to patients with NSCLC. The discovery of the EML4-ALK fusion gene in a limited subset of patients affected by NSCLC and the subsequent clinical development of crizotinib in 2011 has been an impressive milestone in lung cancer research. Unfortunately, acquired resistances regularly develop, hence disease progression occurs. Afterward, modern tyrosine kinase inhibitors (TKIs), such as ceritinib, alectinib, brigatinib, and lorlatinib, have been approved by the Food and Drug Administration (FDA) for the management of anaplastic lymphoma kinase (ALK)-positive NSCLCs. Several compounds are currently under investigation to achieve the optimal strategy of therapy. Additionally, the results of ongoing clinical trials with novel-generation TKI will provide more evidence on the best sequence in the treatment of ALK-positive NSCLC patients. In this review, we provide a comprehensive overview of the state-of-the-art targeted therapy options in ALK-positive NSCLCs. Resistance, potential therapeutic strategies to overcome drug resistance, and future perspectives for this subset of patients are critically analyzed and summarized.
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Wick, Michael J., Elena Helman, Teresa L. Vaught, Monica Farley, Drew W. Rasco, Lon Smith, Anthony Tolcher, Amita Patnaik, Michael Clark, and Kyriakos P. Papadopoulos. "Establishment and Characterization of a Patient-Derived Model of ALK-Positive Anaplastic Large Cell Lymphoma." Blood 126, no. 23 (December 3, 2015): 5121. http://dx.doi.org/10.1182/blood.v126.23.5121.5121.
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Abstract Background: Anaplastic large cell lymphoma (ALCL) is an uncommon, aggressive CD30-positive T-cell lymphoma exhibiting or lacking chromosomal translocation involving the Anaplastic Lymphoma Kinase (ALK) gene and expression of ALK protein. ALK positive ALCL (ALK+-ALCL) typically has an improved prognosis compared with ALK-negative; however, patients often respond favorably to standard chemotherapy and CD30-directed immunoconjugates only to progress and are left with few treatment options. To help identify additional treatment options, we collected tumor tissue and established, characterized and evaluated in vivo an ALK+-ALCL patient-derived xenograft (PDX) model from a thirty one year old Caucasian female with relapsed ALCL following initial therapy with a pediatric S-phase specific regimen. Methods: The ST2698 ALK+-ALCL model was established in CB17 SCID mice using tissue collected from a lymph node tumor biopsy. Clinical tissue, patient blood and the PDX model were subjected to WGS sequencing using an augmented and content-enhanced exome. The augmented exome is optimized to detect major cancer mutations by enhancing coverage over known sequencing gaps and GC-rich regions across >1300 cancer and 200 miRNA genes. We also performed whole-transcriptome sequencing on the PDX model. All data were analyzed using a cancer bioinformatics pipeline optimized for high accuracy detection of small variants and indels, somatic copy-number aberrations, gene expression and fusions. Drug sensitivity studies were performed evaluating sensitivity of the model to patient's current clinical treatment and relevant targeted therapies; study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Exome analysis identified several variants which were confirmed by transcriptome data. Efficacy studies confirmed model sensitivity to the patient's current clinical course (CHOP). In addition, ST2698 was found moderately sensitive to the IMiD lenalidomide (T/C=47%). However, treatment with the ALK-inhibitors crizotinib or ceritinib resulted in tumor regressions including durable complete responses in some mice. Conclusion: We have established and characterized a patient-derived ALK+-ALCL xenograft model using DNA and RNA-based analysis. In addition we evaluated the model in vivo and confirmed sensitivity to the patient's current clinical course and identified two ALK inhibitors as active, including reported complete tumor regressions. Disclosures Rasco: Celgene: Research Funding; Asana BioSciences, LLC: Research Funding. Tolcher:Asana BioSciences, LLC: Consultancy, Research Funding; AbbVie: Consultancy; ArQule: Consultancy; Bayer: Consultancy; BioMed Valley Discoveries: Research Funding; Janssen R&D: Consultancy.
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Tan, Aaron C., and Nick Pavlakis. "Anti-Angiogenic Therapy in ALK Rearranged Non-Small Cell Lung Cancer (NSCLC)." International Journal of Molecular Sciences 23, no. 16 (August 9, 2022): 8863. http://dx.doi.org/10.3390/ijms23168863.
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The management of advanced lung cancer has been transformed with the identification of targetable oncogenic driver alterations. This includes anaplastic lymphoma kinase (ALK) gene rearrangements. ALK tyrosine kinase inhibitors (TKI) are established first-line treatment options in advanced ALK rearranged non-small cell lung cancer (NSCLC), with several next-generation ALK TKIs (alectinib, brigatinib, ensartinib and lorlatinib) demonstrating survival benefit compared with the first-generation ALK TKI crizotinib. Still, despite high objective response rates and durable progression-free survival, drug resistance inevitably ensues, and treatment options beyond ALK TKI are predominantly limited to cytotoxic chemotherapy. Anti-angiogenic therapy targeting the vascular endothelial growth factor (VEGF) signaling pathway has shown efficacy in combination with platinum-doublet chemotherapy in advanced NSCLC without a driver alteration, and with EGFR TKI in advanced EGFR mutated NSCLC. The role for anti-angiogenic therapy in ALK rearranged NSCLC, however, remains to be elucidated. This review will discuss the pre-clinical rationale, clinical trial evidence to date, and future directions to evaluate anti-angiogenic therapy in ALK rearranged NSCLC.
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Gainor, Justin F., Anna M. Varghese, Sai-Hong Ignatius Ou, Sheheryar Kabraji, Mark M. Awad, Ryohei Katayama, Amanda Pawlak, et al. "A retrospective analysis of the prevalence of EGFR or KRAS mutations in patients (pts) with crizotinib-naïve and crizotinib-resistant, ALK-positive non-small cell lung cancer (NSCLC)." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 8083. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.8083.
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8083 Background: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of NSCLC. Recently, several studies have reported that ALK+ pts occasionally harbor concomitant mutations in other oncogenic drivers. Methods: We retrospectively analyzed tumor genotyping data from 1,683 pts with NSCLC seen at 3 U.S. centers from 2009 – 2012 to determine rates of overlapping alterations in EGFR, KRAS and ALK. Mutations in EGFR and KRAS were mainly identified using the SNaPshot multiplexed assay (>95% of cases). ALK FISH was performed in all cases. To determine if this prevalence is impacted by crizotinib, we also updated our earlier analysis (Katayama et al., Sci Transl Med, 2012) of a series of repeat biopsy specimens from 34 crizotinib-resistant, ALK+ pts. Resistant specimens were examined using ALK FISH, SNaPshot, and direct sequencing of the ALK tyrosine kinase domain (TKD). Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. 4 pts with KRAS mutations also had abnormal ALK FISH patterns, involving isolated 5’ green probes (3/4 cases) and an isolated 3’ red probe that was unusually small (1/4 cases). Sufficient tissue was available for confirmatory ALK immunohistochemistry (clone 5A4, Novacastra, UK) in 3 of these cases, all of which were negative for ALK expression. Among pts with ALK+ NSCLC and acquired crizotinib resistance, repeat biopsy specimens remained ALK fusion positive in 28/28 (100%) cases. Secondary mutations in the ALK TKD (1151Tins, L1196M, G1202R, S1206Y, and G1269A) were identified in 7/34 (20.6%) cases. L1196M was the most common secondary mutation (3/34, 8.8% cases). ALK gene amplification was present in 3/28 (10.71%) pts. No EGFR or KRAS mutations were identified in 23 crizotinib-resistant, ALK+ pts with sufficient tissue for testing. Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK+ pts with acquired resistance to crizotinib.
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Witek, Gabriela Maria, Whelton Miller, David Slochower, Esther Berko, Yael Mossé, Mark Lemmon, and Ravi Radhakrishnan. "4558 Investigating the functional consequences of anaplastic lymphoma kinase (ALK) mutations arising upon Lorlatinib treatment." Journal of Clinical and Translational Science 4, s1 (June 2020): 9–10. http://dx.doi.org/10.1017/cts.2020.74.
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OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.
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Ando, Koichi, Ryo Manabe, Yasunari Kishino, Sojiro Kusumoto, Toshimitsu Yamaoka, Akihiko Tanaka, Tohru Ohmori, and Hironori Sagara. "Comparative Efficacy of ALK Inhibitors for Treatment-Naïve ALK-Positive Advanced Non-Small Cell Lung Cancer with Central Nervous System Metastasis: A Network Meta-Analysis." International Journal of Molecular Sciences 24, no. 3 (January 23, 2023): 2242. http://dx.doi.org/10.3390/ijms24032242.
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Central nervous system (CNS) metastases and acquired resistance complicate the treatment of anaplastic lymphoma kinase (ALK) rearrangement-positive (ALK-p) advanced non-small cell lung cancer (NSCLC). Thus, this review aimed to provide a comprehensive overview of brain metastasis, acquired resistance, and prospects for overcoming these challenges. A network meta-analysis of relevant phase III randomized controlled trials was performed to compare the efficacies of multiple ALK inhibitors by drug and generation in overall patients with ALK-p untreated advanced NSCLC and a subgroup of patients with CNS metastases. The primary endpoint was progression-free survival (PFS). Generation-specific comparison results showed that third-generation ALK inhibitors were significantly more effective than second-generation ALK inhibitors in prolonging the PFS of the subgroup of patients with CNS metastases. Drug-specific comparison results demonstrated that lorlatinib was the most effective in prolonging PFS, followed by brigatinib, alectinib, ensartinib, ceritinib, crizotinib, and chemotherapy. While lorlatinib was superior to brigatinib for PFS in the overall patient population, no significant difference between the two was found in the subgroup of patients with CNS metastases. These results can serve as a foundation for basic, clinical, and translational research and guide clinical oncologists in developing individualized treatment strategies for patients with ALK-p, ALK inhibitor-naive advanced NSCLC.
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Yang, Li-Jun, Shuhong Han, Hai Wang, Merry-Jennifer Markham, Brian A. Gray, Westley Reeves, and Lung-Ji Chang. "Characterization and Immunotherapeutic Implication of a Novel Cerebro-Spinal Fluid-Derived ALK-Positive Anaplastic Large Cell Lymphoma Cell Line." Blood 120, no. 21 (November 16, 2012): 5096. http://dx.doi.org/10.1182/blood.v120.21.5096.5096.
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Abstract Abstract 5096 Systemic anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) with characteristic t(2;5) is an aggressive non-Hodgkin lymphoma compared with ALK- ALCL, but has a better prognosis with a 5-year survival of 70–80%. ALK+ ALCL has its unique clinical presentation, pathology, and response to conventional first line CHOP chemotherapy. In general, the treatment induces a complete remission in up to 95% of the patients, but relapse and resistance occur in more than 40% of cases associated with new tumor genetic variants. These ALCL variants have poor prognosis with a tendency of frequent and early relapses. To understand the mechanisms behind treatment resistance and relapse, and to explore new targeted or immune therapy, it is highly desirable to obtain ALK+ALCL cell lines carrying such genetic variants. Using a novel immortalization lentiviral vector system (immLV) expressing multiple cell cycle regulatory genes, we report the establishment of such an ALK+ ALCL line (CSF-LV) derived from cerebrospinal fluid (CSF) of a 25-year-old pregnant woman with relapsing/refractory systemic ALK+ ALCL carrying trisomy 7 and 18 in addition to classic t(2;5). In brief, primary CSF ALK+ ALCL cells were propagated in culture under the following treatment conditions: 1) no immLV, 2) immLV, or 3) immLV plus cytokines. The tumor cells did not expand under condition 1, but expanded into stable cell line under condition 2 with a doubling time around 24 hrs. The CSF-LV cells varied in size from small/medium to large/pleomorphic, and expressed CD30, ALK, CD45, EMA, CD25, HLA-DR, and perforin, but lacked T-cell markers (CD3, CD4, CD5, CD7 and CD8), B-cell markers (CD19, CD20), and myeloid/NK markers (CD14, CD11b and CD56). The CSF-LV cells are clonally rearranged in T-cell receptor genes as illustrated by PCR. The CSF-LV cells have stem genetic lesions t(2, 5)(2p23;5q35) and trisomy 7 and 18 in all examined cells. Subcutaneous injection of the CSF-LV cells into NOD/scid mice demonstrated its tumourigenicity. Histologically, the tumor xenografts displayed morphology and immunohistochemical characteristics similar to the diagnostic specimens of ALK+ ALCL in breast and lymph nodes from the patient. The CSF-LV cells were sensitive to the newly approved ALK inhibitor, Crizotinib, at a concentration of 10 nM; proliferation was blocked around day 4 and apoptosis was detected within 48–72 hours. Interestingly, in condition 3, in the presence of immLV plus cytokines (IL-2 50U/ml, IL-7 20ng/ml and IL-15 20ng/ml), the tumor cells disappeared from the culture at 4 weeks as confirmed by flow cytometry. Furthermore, this was accompanied by an expansion of T cells (CD4+ 86%, CD8+ 12%). CSF-LV tumor-specific T-cell response was demonstrated by the detection of TNFa and IFNg positive T-cells. It is plausible that the ex vivo cytokine-mediated activation/expansion of tumor-specific T cells led to the disappearance of the tumor cells. In summary, we have established a novel ALK+ ALCL cell line from a patient's CSF specimen with aggressive/refractory clinical course. The CSF-LV ALCL cell line has unique genetic signature of t(2, 5)(2p23;5q35) along with trisomy 7 and 18. Despite the refractory clinical course, the tumor cells are very sensitive to targeted ALK inhibitor therapy and the activation of tumor-specific T cells offers new insights into novel immunotherapy for refractory ALCL. Disclosures: No relevant conflicts of interest to declare.
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Hiwatari, Mitsuteru, Kentaro Watanabe, Yasuo Kubota, Shunsuke Kimura, and Junko Takita. "Identification of Novel Pathways and Small Molecules Able to Down Regulate Oncogenes Expression By Compounds Screening Approaches in Acute Leukemia and Neuroblastoma Cells." Blood 132, Supplement 1 (November 29, 2018): 2214. http://dx.doi.org/10.1182/blood-2018-99-111090.
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Abstract Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, resistance to ALK inhibitors is a ubiquitous problem in ALK-expressing cell lines as well as treated patients. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. Previous reports have suggested that mechanism of resistance is mediated by mutations in the ALK kinase domain impairing binding of an inhibitor to an ALK protein. Thus, new treatment modalities are urgently needed to sensitize patients to crizotinib thereby improving the management of hematological or solid malignancies harboring ALK mutations. To identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, we implemented a high throughput chemical screen in 4 NB-derived cell lines, using a curated library of ~450 compounds. In the compounds screening, JAK-STAT kinase inhibitor (cucurbitacin I) was the most discriminatory with regard to sensitivity for ALK-mutated cell lines. Since Gamma cytokine JAK/STAT system as a target in the treatment of T-cell or myeloid malignancies, we analyzed the cytotoxicity of cucurbitacin I antitumor effect using an MTT assay revealed cucurbitacin I to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by cucurbitacin I (IC50values:T-ALL lines, ICH-TALL-UK, 103.2 nM; MOLT-14, 35.6 nM; KCMC-T, 134.2 nM; Jurkat, 77 nM; ICH-TALL-SM, 47.7 nM; MOLT-4, 22.2 nM. AML lines, HEL, 66.7 nM; Kasumi-3, 4.5 nM; KG-1, 53.6 nM; THP-1, 337.2 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of cucurbitacin I. In NB cell lines harboring F1174L or R1275Q-mutated ALK, crizotinib combined with cucurbitacin I enhanced tumor responses and showed synergistic cytotoxicity.Although crizotinib and cucurbitacin I alone or combination therapy (cucurbitacin I + crizotinib) did not result in decreased viability over control compared with vehicle, the combination therapy in all of 6 cell lines with ALK aberrations and 10 of 13 ALK wild-type cell lines with MYCN amplification or 11q LOH was more effective than vehicle, crizotinib alone, and cucurbitacin I alone. Analysis of downstream signalling through MAPK, AKT and STAT3 pathways showed that NIH3T3 cells stably expressed F1174L mutated ALK or TGW cells harboring R1275Q-mutated ALK, expressed lower levels of pERK, pAKT and pSTAT3 in combination therapy compared with cells treated with cucurbitacin I or crizotinib alone. These findings may provide a indication that the combination of low-dose ALK and STAT3 inhibitors may be benefitical for the treatment of NB, by enhancing efficacy while reducing toxicity. In conclusion, our studies suggested that NB, T-ALL or AML cell lines also exhibited potent cytotoxic responses to the cucurbitacin I and the combination of ALK and JAK-STAT inhibitors could be a valuable therapeutic option for ALK mutated malignancies such as high-risk NB with potential clinical application. Disclosures No relevant conflicts of interest to declare.
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Das, Arabinda, Ron Ron Cheng, Megan L. T. Hilbert, Yaenette N. Dixon-Moh, Michele Decandio, Alex Vandergrift William, Naren L. Banik, et al. "Synergistic Effects of Crizotinib and Temozolomide in Experimental FIG-ROS1 Fusion-Positive Glioblastoma." Cancer Growth and Metastasis 8 (January 2015): CGM.S32801. http://dx.doi.org/10.4137/cgm.s32801.
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Glioblastoma (GB) is the most common malignant brain tumor. Drug resistance frequently develops in these tumors during chemotherapy. Therefore, predicting drug response in these patients remains a major challenge in the clinic. Thus, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Robust experimental evidence has shown that the main reason for failure of treatments is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK), c-Met (hepatocyte growth factor receptor), and oncogenic c-ros oncogene1 (ROS1: RTK class orphan) fusion kinase FIG (fused in GB)-ROS1. As such, these could be attractive targets for GB therapy. The study subjects consisted of 19 patients who underwent neurosurgical resection of GB tissues. Our in vitro and ex vivo models promisingly demonstrated that treatments with crizotinib (PF-02341066: dual ALK/c-Met inhibitor) and temozolomide in combination induced synergistic antitumor activity on FIG-ROS1-positive GB cells. Our results also showed that ex vivo FIG-ROS1+ slices (obtained from GB patients) when cultured were able to preserve tissue architecture, cell viability, and global gene-expression profiles for up to 14 days. Both in vitro and ex vivo studies indicated that combination blockade of FIG, p-ROS1, p-ALK, and p-Met augmented apoptosis, which mechanistically involves activation of Bim and inhibition of survivin, p-Akt, and Mcl-1 expression. However, it is important to note that we did not see any significant synergistic effect of crizotinib and temozolomide on FIG-ROS1-negative GB cells. Thus, these ex vivo culture results will have a significant impact on patient selection for clinical trials and in predicting response to crizotinib and temozolomide therapy. Further studies in different animal models of FIG-ROS1-positive GB cells are warranted to determine useful therapies for the management of human GBs.
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Zhang, Wenqun, Shan Wang, Jing Yang, Bo Hu, Ying Liu, Ling Jing, Qing Zhu, et al. "ALK Inhibitor Plus Vinblasitine for Refractory/Relapsed Pediatric ALK+ Anaplastic Large Cell Lymphoma: A Prospective, One-Arm, Open-Label Real-World Study." Blood 138, Supplement 1 (November 5, 2021): 1406. http://dx.doi.org/10.1182/blood-2021-149402.
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Abstract Background: Refractory and relapsed (r/r) anaplastic lymphoma kinase (ALK)-positive anaplastic large cell Lymphoma(ALK+ALCL) have a poor prognosis. Studies had confirmed vinblastine (VBL) may be an effective treatment for relapsed ALCL and ALK inhibitors may have high efficacy and tolerability in patients carrying ALK fusions. Therefore, we put forward a hypothesis that for those r/r ALK+ALCL patients with poor chemotherapy tolerance and effect, combining ALK inhibitors with vinblastine may have a sustained anti-tumor effect and good tolerance. Aims: To assess the efficacy and safety of ALK inhibitors combined with VBL in pediatric relapsed/refractory (r/r) ALK+ ALCL. Methods: Patients aged between 0 and 18 years with r/r ALK+ ALCL were included in this trial. ALK inhibitors are given orally with a converted dosage according to body surface area and VBL was given with injection once a week at 4-6 mg/(m2.week). And no intensive chemotherapy and hematopoietic stem cell transplantation will be in our study.We may decrease the dosage of VBL or delete VBL if patients suffer severe bone marrow suppression, repeated infections, and peripheral neuritis and other VBL-related toxicities. Available ALK inhibitors included crizotinib(CZ), alectinib(AL) and ceritinib(CER). Patients without CNS involvement took CZ, and patients with CNS involvement took AL or CER. A cycle of therapy is 28 days. Evaluation for responses toxicities were monitored and recorded. Results:27 patients were enrolled in this trial between April 2018 and April 2021. The median age was 8.3 years old(ranging from 0.75 to 16), male/female ratio is19:8. By St.jude's staging, 20 cases were in stage III (74.1%), 7 cases in stage IV (25.9%) including 3 cases (11.1%) with CNS involvement at initial diagnosis. Before being enrolled in our study, 6 patient (22.2%) failed to achieve CR with prior 4-10 chemotherapy regimens, 21 patients (77.8%) relapsed after an average of 5-16 courses of standard pulse chemotherapy regimens. After 1st cycle of treatments, 62.9%(17/26) achieved comlpele remission(CR) without measurable tumors and ALK gene transcription, no progressed cases. Additional 6 case got CR after 2-10 month(23.1%). In general, 7 patients withdraw due to death, progress, ineffectiveness, economics, adverse reactions. The treatment retention rate was ,7.1%（20/7），objective response rate (ORR) was 85.2%(23/27), best complete remission rate(CRR) was 85.2%(23/27), disease control rate(DCR) was 92.5%(25/27), average duration of CR was 14.85 months(0-36months). Toxicities included nausea and vomiting, stomachache, diarrhea(81.5%,22/27, one or more above toxicities), neutropenia (27/27), pneumonia(7.4%, 2/27), sepsis(7.4%, 2/27), creatine kinase-MB elevation (81.5%,22/27,all are in crizotinib group including 2 abnormal ECG cases), liver enzyme elevation (63.%,17//27), intestinal obstruction(7.4%, 2/26). 2 cases relapsed because of the treatment discontinuation due to severe elevated liver enzymes. Summary/Conclusion: ALK inhibitors combined with VBL may rapidly induce the tumor remission of r/r ALK+ALCL and the tolerance is good. Combination therapy may induce a more lasting remission in for patients without BM involvement. Patients with BM involvement are more likely to fail treatment. Sequential use of ALK kinase inhibitors may be an efficient strategy to counter drug resistance.More data and longer follow-up time are needed to support our study. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Elsayed, Mei, and Petros Christopoulos. "Therapeutic Sequencing in ALK+ NSCLC." Pharmaceuticals 14, no. 2 (January 21, 2021): 80. http://dx.doi.org/10.3390/ph14020080.
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Anaplastic lymphoma kinase-rearranged non-small-cell lung cancer (ALK+ NSCLC) is a model disease for the use of targeted pharmaceuticals in thoracic oncology. Due to higher systemic and intracranial efficacy, the second-generation ALK tyrosine kinase inhibitors (TKI) alectinib and brigatinib have irrevocably displaced crizotinib as standard first-line treatment, based on the results of the ALEX and ALTA-1L trials. Besides, lorlatinib and brigatinib are the preferred second-line therapies for progression under second-generation TKI and crizotinib, respectively, based on the results of several phase II studies. Tissue or liquid rebiopsies at the time of disease progression, even though not mandated by the approval status of any ALK inhibitor, are gaining importance for individualization and optimization of patient management. Of particular interest are cases with off-target resistance, for example MET, HER2 or KRAS alterations, which require special therapeutic maneuvers, e.g., inclusion in early clinical trials or off-label administration of respectively targeted drugs. On the other hand, up to approximately half of the patients failing TKI, develop anatomically restricted progression, which can be initially tackled with local ablative measures without switch of systemic therapy. Among the overall biologically favorable ALK+ tumors, with a mean tumor mutational burden uniquely below 3 mutations per Mb and the longest survival among NSCLC currently, presence of the EML4-ALK fusion variant 3 and/or TP53 mutations identify high-risk cases with earlier treatment failure and a need for more aggressive surveillance and treatment strategies. The potential clinical utility of longitudinal ctDNA assays for earlier detection of disease progression and improved guidance of therapy in these patients is a currently a matter of intense investigation. Major pharmaceutical challenges for the field are the development of more potent, fourth-generation TKI and effective immuno-oncological interventions, especially ALK-directed cell therapies, which will be essential for further improving survival and achieving cure of ALK+ tumors.
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Wu, Rui, Elena Ivan, Anagh Anant Sahasrabuddhe, Timothy Shaw, Charles G. Mullighan, Vasiliki Leventaki, Kojo S. J. Elenitoba-Johnson, and Megan S. Lim. "Epigenetic Modulation of CD48 By NPM-ALK Promotes Immune Evasion in ALK+ ALCL." Blood 134, Supplement_1 (November 13, 2019): 1510. http://dx.doi.org/10.1182/blood-2019-127453.
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Background: NPM-ALK is the prototypical oncogenic fusion characteristic of anaplastic lymphoma kinase anaplastic large cell lymphoma (ALK+ ALCL). While NPM-ALK promotes lymphomagenesis through deregulation of multiple of cellular processes, its role in immune evasion is largely unknown. Significantly, 30% of ALK+ ALCL patients develop resistance to current treatment. Therefore, the identification and development of novel immune modulatory therapies remains an opportunity. Using unbiased mass spectrometry-based N-glycoproteomic analysis, we identified preferential loss of proteins (CD48, CD2 and CD244) that regulate cell-mediated target cell lysis in five ALK+ ALCL cell lines compared with other T cell lymphoma-derived cell lines. We hypothesized that NPM-ALK deregulates the expression of key cell surface molecules that function in immune evasion via an epigenetic mechanism to attenuate nature killer (NK) cell-mediated cytotoxicity against neoplastic ALK+ ALCL cells. Methods: We performed global N-glycoproteomic analysis using a hydrazide-based peptide enrichment chemistry followed by tandem mass spectrometry. We also examined CD48 expression in 5 ALK+ ALCL cell lines by qRT-PCR, flow cytometry and immune-blotting (IB). Immunohistochemistry was used to assess the expression of CD48 in tissue microarray (TMA) of ALK+ ALCL, Peripheral T Cell Lymphoma Not Otherwise Specified (PTCL, NOS) (n=8 and 69 respectively). Cases with more than 15% positive neoplastic cells were scored as positive for CD48. RNA-seq-based gene expression study of 32 ALK+ALCL patients was used to assess the correlation between expression of ALK and CD48, CD2, CD58 and CD244. To assess the role of NPM-ALK in CD48 downregulation in ALK+ ALCL, we stably expressed NPM-ALK in primary CD4+T cells (NA119 and NA149). CD48 promoter methylation was correlated with ALK expression in 27 ALK+ALCL patients by RNA-seq and DNA methylation arrays. The interaction between STAT3 and CD48 promoter was examined by chromatin immunoprecipitation (ChIP). We assessed the effect of Crizotinib (ALK inhibitor), S31-201 (STAT3 inhibitor) and 5-aza-2' deoxycytidine (DNMT1 inhibitor) on the expression of CD48 and attendant NK cell-mediated cytotoxicity using WST-1 assay on ALK+ ALCL cell lines. To assess the functional significance of CD48, ALK+ ALCL cell lines (DEL, Karpas299) were stably transduced with CD48 and NK-cell mediated cytotoxicity was determined. Results: We confirmed the aberrant loss of CD48 in 5 ALK+ ALCL cell lines through qRT-PCR, flow cytometry and IB. In contrast, activated peripheral blood T cells expressed abundant CD48 by IB. In TMAs, ALK+ ALCL demonstrated significantly higher percentage of CD48 negative cases (62.35%) compared with PTCL, NOS (8.7%, p<0.001). Gene expression analysis of ALK+ALCLs showed a negative correlation between ALK and CD48 (R=-0.5537), CD2 (R=-0.5381), and CD244 (R=-0.4177) but not CD58 (R=0.0064). Ectopic expression of NPM-ALK in primary CD4+T cells led to downregulation of CD48. Inhibition of ALK (Crizotinib), STAT3 (S31-201) as well as DNMT1 (5-aza-2'-deoxycytidine) increased CD48 transcript and protein levels in Crizotinib sensitive (Karpas299, NA149) ALK+ ALCL cell lines. DNA methylation analysis of ALK+ ALCL tissue samples showed that the methylation status of the CD48 promoter (1500bp upstream of transcriptional start site) was positively correlated with ALK levels (R= 0.60). ChIP analysis demonstrated that STAT3 directly binds to the CD48 promoter (at 445-0 bp upstream of CD48 transcriptional starting site). NK cell-mediated cytotoxicity against malignant cells was also increased following ALK (DMSO 22.75% vs Crizotinib 55.9%, p=0.0009) and STAT3 (DMSO 22% vs S31-201 55.1%, p<0.001) inhibition. Exogenous expression of CD48 in DEL and Karpas299 cells led to significant increase in the percentage of NK cell-mediated cytotoxicity (DELEV 20.7% vs DELCD48 32.7%, p<0.0313; Karpas299EV 15.5% vs Karpas299CD48 26.9%, p<0.0055). Conclusion: Expression of cell surface molecules such as CD48, CD2 and CD244 that function in immune surveillance is negatively regulated by NPM-ALK in a kinase-dependent manner and lead to immune evasion. Further, the loss of CD48 expression in ALK+ ALCL is mediated by epigenetic mechanisms which maybe reversible and have therapeutic significance in ALK+ ALCL. Disclosures Mullighan: AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.
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Chattopadhyay, Nibedita, Sakeena Syed, Yuhong Zhang, Jie Yu, Dan He McDougall, Lunyin Yu Yu, Stephen Tirrell, et al. "Ninlaro (ixazomib) and Brentuximab Vedotin (ADCETRIS) Combination Results in Synergistic Antitumor Activity in Mouse Models of CD30 Positive Anaplastic Large Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 1842. http://dx.doi.org/10.1182/blood.v128.22.1842.1842.
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Abstract The ubiquitin-proteasome system (UPS) represents the primary mechanism by which cells degrade proteins. Proteasome inhibition results in accumulation of proteasome substrates, leading to cell cycle disruption, endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and, ultimately, the activation of apoptotic pathways and cell death. Ixazomib (NINLARO) is the first FDA-approved oral proteasome inhibitor, indicated in combination with lenalidomide and dexamethasone for treatment of patients with multiple myeloma who have received at least one prior therapy. Ixazomib has been evaluated as a single agent in relapsed/refractory lymphoma, but additional data is needed to identify combination agents for lymphoma trials. Brentuximab vedotin (ADCETRIS) is an anti-CD30 antibody drug conjugate (ADC) that is protease cleavable linker with monomethyl auristatin E (MMAE), a highly potent antimicrotubule agent. Brentuximab vedotin binds specifically to CD30, a cell surface marker expressed at high levels on Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) tumor cells and induces cell death by direct cytotoxicity and also antibody-dependent cellular phagocytosis (ADCP). Brentuximab vedotin is approved in patients with relapsed/refractory HL, relapsed/refractory ALCL, and for consolidation in in the post autologous stem cell transplant (ASCT) setting of HL. Preclinical evidence suggests that resistance to anti-CD30 therapy can be mediated via activation of the NFkB pathway, which can be reversed by proteasome inhibitor treatment, suggesting a rationale for the combination of ixazomib and brentuximab vedotin. In the current study we evaluated this combination in three CD30+ ALCL xenograft models, SR-786, Karpas-299 and SUDHL-2 grown in immunocompromised mice. In SR-786 and Karpas-299, the combination treatment using dose levels of ixazomib and brentuximab vedotin with minimal single-agent activity resulted in synergistic effect with complete tumor regression. In most animals, the tumor did not regrow as long as 60 days after the last dose of combination drug treatment. Unlike in SR-786 and Karpas-299, no combination benefit was observed in the SUDHL-2 xenograft model. Pharmacodynamic studies are ongoing to understand the mechanism of synergy. Our data suggest that combination of ixazomib and brentuximab vedotin warrants clinical evaluation in CD30+ lymphoma patients. Disclosures Chattopadhyay: Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Zhang:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. He McDougall:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Berger:Takeda Pharmaceuticals: Employment. van de Velde:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Tremblay:Takeda Pharmaceuticals: Employment. Huebner:Takeda Pharmaceuticals International Co.: Employment, Equity Ownership.
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Wang, Peng, Moinul Haque, Jing Li, Yung-Hsing Huang, Meaad Almowaled, Carter Bargar, Adam Karpf, Will Chen, Suzanne Turner, and Raymond Lai. "FOXM1 and the NPM-ALK/STAT3 Axis Form a Novel Positive Feedback Loop in Promoting the Oncogenesis of ALK-Positive Anaplastic Large Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 3921. http://dx.doi.org/10.1182/blood-2018-99-115743.
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Abstract Peng Wang1, Moinul Haque2, Jing Li2,3, Yung-Hsing Huang2, Meaad Almowaled2, Carter Bargar4, Adam Karpf4, Will Chen2, Suzanne Turner5 and Raymond Lai2,6 1Division of Hematology, Dept of Medicine, University of Alberta, Edmonton, 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; 3Electron Microscopy Center, Basic Medical Science College, Harbin Medical University, Harbin, Heilongjiang, China; 4Eppley Institute and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, USA; 5Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK;6Department of Oncology, University of Alberta, Edmonton, Alberta, Canada 1. Backgrounds and Aims Forkhead Box M1 (FOXM1) is a transcription factor implicated in the pathogenesis of solid tumors, and it has been shown to promote cell-cycle progression, stem cell renewal and chemotherapeutic resistance in cancer cells. Nonetheless, the biological significance of FOXM1 in hematologic malignancies has not been extensively studied. Here, we studied the expression and role of FOXM1 in ALK-positive anaplastic large cell lymphoma (ALK+ALCL). 2 Methods and Results In contrast with normal lymphocytes, FOXM1 was highly expressed in all ALK+ALCL cell lines (5/5), tumors from patients (6/6) and tumors arising from NPM-ALK transgenic mice. Experiments using nuclear/cytoplasmic fractionation, immunocytochemistry and reporter assays had provided evidence that FOXM1 is transcriptionally active in ALK+ALCL. Down-regulation of FOXM1 expression using shRNA and a pharmacologic agent (thiostrepton) resulted in a significant reduction in cell growth, colony formation in soft agar and cell-cycle arrest in ALK+ALCL cells. Further studies revealed that the oncogenic potential of FOXM1 is linked to substantial increases in the phosphorylation/activation status of NPM-ALK and STAT3, and the upregulations of a host of cytokines that have been previously shown to activate the NPM-ALK/STAT3 axis, including IGF-1, IL9 and IL21. Using co-immunoprecipitation, we found that NPM-ALK binds to FOXM1 in the nucleus of ALK+ALCL cells. Importantly, the binding of NPM-ALK to FOXM1 promotes the DNA binding ability and transcriptional activity of FOXM1, and functional inhibition of NPM-ALK using crizotinib or depletion of NPM-ALK using siRNA in ALK+ALCL cells significantly decreased the transcriptional activity of FOXM1.Conclusions: In conclusion, we have identified a novel oncogenic feedback loop involving FOXM1 and the NPM-ALK/STAT3 axis in ALK+ALCL. This study has revealed the first clear example in which NPM-ALK exerts important oncogenic functions in the nuclei of ALK+ALCL cells, by means of its binding to an oncogenic transcription factor so as to promote its DNA binding and transcription activity. Disclosures No relevant conflicts of interest to declare.
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Ducray, Stephen P., Ricky Trigg, Andrew J. Bannister, Raymond Lai, Gerda Egger, Olaf Merkel, Lukas Kenner, and Suzanne Turner. "The Identification of Novel Epigenetic Therapies for ALK-Driven Haematological Malignancies." Blood 134, Supplement_1 (November 13, 2019): 1483. http://dx.doi.org/10.1182/blood-2019-125648.
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Introduction Through conserved signalling pathways, Anaplastic Lymphoma Kinase (ALK) is well-described in driving haematological malignancies including Anaplastic Large Cell Lymphoma (ALCL) and Diffuse Large B-Cell Lymphoma (DLBCL) and as such presents itself as an amenable therapeutic target. Hence, directed therapeutics (ALK tyrosine kinase inhibitors; TKI) are being used in the treatment of ALK-driven cancers. Unfortunately, findings in the clinic and clinical research studies have taught us that resistance to ALK inhibitors can develop through the activation of ALK signalling bypass tracks. As such there is a need for the development of novel front-line, dual-combination, as well as second-line therapies. Methods A large-scale epigenetic targeted drug library consisting of approximately 300 FDA-approved drugs and novel agents was applied to a number of cell lines representing ALK-driven haematological malignancies: ALCL cell lines (DEL, JB-6, KARPAS-299, SU-DHL-1, SUP-M2) and the DLBCL cell line LM-1. Drugs which caused a >75% decrease in cell viability were classified as 'candidate drugs' and studied further. Results Several of the validated drugs have previously been used/trialled in the clinic for the treatment of various cancers, e.g. aurora kinase (XL-228), topoisomerase (Mitoxantrone HCl) and HDAC (Romidepsin) inhibitors - these functioned as internal controls for the drug screens. However, an assortment of novel drugs was also identified that have not previously been described in the context of the treatment of ALK-driven haematological malignancies; including the FLT3 inhibitor KW2449 which caused a >75% decrease in viability in all the tested cell lines and as such may serve as a novel front line therapy. In addition, a novel DNA methyltransferase (DNMT) inhibitor was identified which is efficacious and resulted in a >90% decrease in viability in all cell lines treated across both disease entities. Furthermore, we investigated the combinatorial potential of the identified DNMT inhibitor with ALK TKIs such as Brigatinib and observed the inhibitor acting synergistically (as per Bliss-Independence calculations) resulting in a further decrease in cell viability. Several cell lines that are resistant to ALK TKIs were also assessed for their sensitivity to the DNMT inhibitor and were shown to be susceptible to this drug, as demonstrated by a significant decrease in cell viability. Figure 1: (A) Viability following drug screen in a representative cell line. Those drugs which led to a >75% change in viability were taken forward for validation, as shown in (B). (C) Candidate drugs identified for each of the cell lines tested, grouped according to their molecular target. Conclusion In conclusion, an epigenetic drug library has been employed to identify novel therapeutic agents for the treatment of ALK-driven haematological malignancies including ALCL and DLBCL. Data reveal a potent inhibitor of DNA methylation as a candidate drug that suppresses the growth of ALK-driven malignancies both alone and in combination with ALK TKIs. Significantly, this identified drug also inhibits the growth of cell lines resistant to directed therapeutics such as ALK TKIs suggesting it has potential clinical use. Disclosures No relevant conflicts of interest to declare.
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Ceccon, Monica, Mario Mauri, Luca Massimino, Giovanni Giudici, Rocco Piazza, Carlo Gambacorti-Passerini, and Luca Mologni. "Mitochondrial Hyperactivation and Enhanced ROS Production are Involved in Toxicity Induced by Oncogenic Kinases Over-Signaling." Cancers 10, no. 12 (December 12, 2018): 509. http://dx.doi.org/10.3390/cancers10120509.
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Targeted therapy is an effective, rational, and safe approach to solid and hematological tumors treatment. Unfortunately, a significant fraction of patients treated with tyrosine kinase inhibitors (TKI) relapses mainly because of gene amplification, mutations, or other bypass mechanisms. Recently a growing number of papers showed how, in some cases, resistance due to oncogene overexpression may be associated with drug addiction: cells able to proliferate in the presence of high TKI doses become also TKI dependent, undergoing cellular stress, and apoptosis/death upon drug withdrawal. Notably, if a sub-cellular population survives TKI discontinuation it is also partially re-sensitized to the same drug. Thus, it is possible that a subset of patients relapsing upon TKI treatment may benefit from a discontinuous therapeutic schedule. We focused on two different hematologic malignancies, chronic myeloid leukemia (CML) and anaplastic large cell lymphoma (ALCL), both successfully treatable with TKIs. The two models utilized (LAMA and SUP-M2) differed in having oncogene overexpression as the sole cause of drug resistance (CML), or additionally carrying kinase domain mutations (ALCL). In both cases drug withdrawal caused a sudden overload of oncogenic signal, enhanced mitochondria activity, induced the release of a high amount of reactive oxygen species (ROS), and caused genotoxic stress and massive cell death. In LAMA cells (CML) we could rescue the cells from death by partially blocking downstream oncogenic signaling or lowering ROS detrimental effect by adding reduced glutathione.
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Gaudio, Eugenio, Chiara Tarantelli, Alberto Arribas, Luciano Cascione, Ivo Kwee, Andrea Rinaldi, Maurilio Ponzoni, et al. "Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529." Blood 128, no. 22 (December 2, 2016): 4187. http://dx.doi.org/10.1182/blood.v128.22.4187.4187.
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Abstract Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response. Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations. We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC. Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.
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Chen, Robert, Jessie Hou, Edward Newman, Young Kim, Cecile Donohue, Stephen J. Forman, and Susan Kane. "Downregulation of CD30, Resistance to MMAE, and Upregulation of MDR1 Are All Associated with Resistance to Brentuximab Vedotin." Blood 124, no. 21 (December 6, 2014): 3643. http://dx.doi.org/10.1182/blood.v124.21.3643.3643.
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Abstract Background: Both Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) express surface CD30. Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. Although BV elicits a high response rate (75% in HL and 86% in ALCL), the majority of patients who do not attain complete response (CR) will eventually develop resistance to BV. It is not known whether resistance to BV is through a) CD 30 alterations b) resistance to cytotoxic agent MMAE or c) overexpression of drug exporters. We developed 2 BV-resistant cell models and obtained primary lymphoma samples from patients with relapsed/progressive disease post BV therapy. We examined CD30 expression, MMAE resistance, drug exporter expression, and gene expression profiles in vitro and in vivo to determine mechanisms of resistance to BV. Methods: HL cell line(L428) and ALCL cell line (KARPAS 299) were used for in vitro experiments. The selection of BV resistant cell model (L428R and KARPAS 299R) used two different approaches (pulsatile or constant exposure). Both BV resistance and MMAE resistance were confirmed by MTS assays. CD30 expression was measured by flow cytometry,qRT-PCR, and Western blotting. Drug exporter expression was measured using qRT-PCR to MDR1, MRP1, and MRP3. In vivo experiments utilized primary tumor samples from 15 HL and 4 ALCL patients who had developed relapsed/progressive disease post BV treatment. CD30 expression was assessed by immunohistocytochemistry (IHC). Gene expression profiling was performed in both parental and resistant HL and ALCL cells, and in 4 ALCL primary tumor samples using Affymetrix whole genome GeneChip® Human Genome U133 2.0 Plus. Results: MTS assay showed the IC50 of KARPAS 299R to BV shifted from 24 +/- 10 ng/ml to 28 +/- 9 ug/ml, an 1183-fold increase. MTS assay also showed the IC50 of KARPAS 299R to MMAE only increased 2-fold when compared to KARPAS 299. Flow cytometry showed downregulation of surface CD30 expression in KARPAS 299R as compared to KARPAS 299 parental (59% vs. 96%, median intensity 78 +/- 17 vs. 591 +/- 51). This downregulation was confirmed by qRT-PCR and Western blotting for CD30. As KARPAS 299R is a mixed cell population, we sorted them into CD30+ and CD30- subpopulations. We then analyzed for BV sensitivity based on CD 30 expression status in KARPAS 299R. MTS assay showed that KARPAS 299R CD30+ cells were equally as resistant to BV as KARPAS 299R CD30- cells (figure 1A). IHC performed in 4 ALCL primary tumor samples showed persistent CD 30 expression in relapsed/progressive tumor specimens post BV treatment. Gene expression profiling on KARPAS 299R showed downregulation of CD30 as compared to KARPAS 299. Gene expression profiling on pre- and post-treatment ALCL samples (8) did not show significant differences in CD30 expression. The top four upregulated genes in relapsed/progressive samples as compared to pretreatment samples were LCE3D, WNT3, TNNT, CITED2. The top four downregulated genes in relapsed/progressive samples as compared to pretreatment samples were CXCL13, C4orf7, MS4A1, and IGJ. MTS assay showed that the IC50 of L428R to BV has shifted from 32 +/- 11 ug/ml to 391 +/- 92 ug/ml, a 12-fold increase. MTS assays showed the IC50 of L428R to MMAE has increased 99-folds when compared to L428 (figure 1B). No difference was seen in CD 30 expression by flow cytometry, qRT-PCR, or western blotting between L428R vs. L428. IHC performed in 15 HL primary tumors show persistent CD30 expression in relapsed/progressive tumor specimen post-BV treatment. qRT-PCR showed upregulation of MDR1mRNA in L428R as compared to L428. Gene expression profiling on L428R showed upregulation of MDR1 as compared to L428. Conclusion: Downregulation of CD30 is seen in BV-resistant ALCL cell model. However, sensitivity to BV did not depend solely on the level of CD30 expression as CD30+ cell subpopulations still exhibited resistance to BV in vitro. Upregulation of MDR-1 and resistance to MMAE were seen in BV-resistant HL cells, rather than downregluation of CD30. Downregulation of CD30 was not seen in HL or ALCL primary tumors. Further work is ongoing to explore/validate potential targets derived from gene expression profiling in ALCL primary tumors. Figure 1A Sensitivity to BV is not related to CD30 expression Figure 1A. Sensitivity to BV is not related to CD30 expression Figure 1B. Figure 1B. Viability of L-428 parental versus BV-resistant cells Disclosures Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Travel expenses Other.
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Chen, Robert, Jessie Hou, Megan Reddy, Edward Newman, Stephen J. Forman, and Susan Kane. "Selection For Brentuximab Vedotin Resistant Lymphoma Cell Lines Leads To Downregulation Of Surface CD30 Expression." Blood 122, no. 21 (November 15, 2013): 1280. http://dx.doi.org/10.1182/blood.v122.21.1280.1280.
Full textAbstract:
Abstract Background Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. In pivotal phase II trials BV demonstrated a complete response rate (CR) of 34% in patients with relapsed/refractory Hodgkin lymphoma (HL) and CR of 50% in patients with relapsed/refractory anaplastic large cell lymphoma (ALCL). However, patients who do not attain CR will eventually develop progressive disease despite active treatment with BV. We aim to select for BV resistant lymphoma cell lines and examine for alterations in surface CD30 expression in cell lines. Methods In vitro experiments used two HL cell lines (L428, KM-H2) and one ALCL cell line (Karpas 299). The selection of BV resistant cell lines was done in two different approaches (pulsatile and constant exposure). Confirmation of BV resistance was done by MTS assays and cell counting assays. Measurement of surface CD30 was performed by flow cytometry using monoclonal mouse anti-human CD 30, clone Ber-H2. Results MTS assay showed the IC50 of parental cell lines to be: L428 (24 ug/ml), KM-H2 (9 ug/ml), and Karpas (16 ng/ml). The cell lines were incubated at supra-IC50 dosage until no proliferation was seen and then rescued with BV free media in the pulsatile approach. Only L428 was able to be rescued and kept in BV free media until consistent proliferation was seen (defined as incremental growth for 3 consecutive weeks). L428 was then incubated again in 50 mg/ml of BV until consistent proliferation was seen. For the constant exposure approach, the cell lines were incubated at sub-IC50 dosages and dosages of BV were adjusted up depending on cell proliferation rates. Only KM-H2 and Karpas 299 showed consistent proliferation in the constant exposure approach. We were able to select for 1 resistant line per cell type. Cell counting assay showed that all three resistant cell lines were able to proliferate in the presence of BV at dosages twice their respective IC50s (Figure 1, only L428 is shown). MTS assays showed that all resistant cell lines had increased IC50s (Table 1). Flow cytometry showed downregulation of surface CD30 expression in Karpas 299 and KM-H2 but not L428 (Table1). For Karpas 299, both the percentage of CD30+ cells and the median CD30 intensity decreased in the resistant line. For KM-H2, only the median CD30 intensity decreased in the resistant line. Conclusion Prolonged exposure of HL and ALCL cell lines to BV led to resistant cell lines. When compared to their parental cell lines, two out of three resistant lines showed downregulation in surface CD30 expression. Disclosures: Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Trave expenses Other.
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Friday, Ellen, Gang Ye, and Francesco Turturro. "Study of the Combination of Bortezomib with PPAR/RXR Agonists Rosiglitazone and Bexarotene in Lymphoproliferative Disorder-Derived Cell Linesγ." Blood 106, no. 11 (November 16, 2005): 4427. http://dx.doi.org/10.1182/blood.v106.11.4427.4427.
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Abstract It has been recently shown that the combination of peroxisome proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR) ligands enhances their activity as inducers of differentiation and apoptosis. It has also been demonstrated that the induction of the ubiquitin-dependent degradation of PPARγ/RXR heterodimers by the proteosome is activated upon binding of a receptor ligand. Based on this knowledge, we hypothesized that the use of a proteosome inhibitor may enhance the activity of the receptor ligands by prolonging their activation. For this purpose, we tested the antiproliferative activity of the combination of the proteosome inhibitor, bortezomib with PPARγ and RXR ligands, rosiglitazone, and bexarotene as compared to single agents in a series of cell lines derived from ALK+ (JB6, SUPM2)/ALK- (SKB, MAC1) anaplastic large cell lymphoma (ALCL), Burkitt’s lymphoma (Hs-Sultan) and multiple myeloma (ARH77). In the combination studies, 5 nM bortezomib was added to cell culture 6 h prior to the addition of 10 μM rosiglitazone and/or 25 μM bexarotene. None of the cell lines reached IC50 as assessed by CellTiter 96 Aqueous cell proliferation assay (Promega), and represented as percent of untreated controls, following 72 h incubation with serial concentrations of rosiglitazone (5, 10, 25, 50 μM) or bexarotene (25 and 50 μM). Bortezomib IC50 was reached only in JB6 cells at 5 nM or in MAC1 and SKB cells at 20 nM at 72 h. The IC50s were reached by 5 nM bortezomib/10 μM rosiglitazone in SUPM2 cells or by 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene in Hs-Sultan cells at 72 h. Multiple myeloma-derived cells ARH77 showed resistance to single agent bortezomib, rosiglitazone or bexarotene at all the concentrations tested. However, in these cells, IC50 was reached only by the combination of 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene at 96 h of incubation. Surprisingly, ALK+ALCL-derived JB6 cells reached their IC50s with any of the drug combinations (5 nM bortezomib/10 μM rosiglitazone; 5 nM bortezomib/25 μM bexarotene; 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene) between 24 (5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene) and 72 h (5 nM bortezomib/10 μM rosiglitazone; 5 nM bortezomib/25 μM bexarotene). After 72 h of incubation, the percentage of JB6 viable cells treated with 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene as compared to untreated controls, was significantly lower than the percentage of cells treated with single agent 5 nM bortezomib (22% ±0.6 vs 45% ± 1.4, P<0.05). In conclusion, our data show that bortezomib at the lowest IC50 (5 nM/JB6 cells) enhances the antiproliferative effects of rosiglitazone or bexarotene in JB6, SUPM2 and Hs-Sultan cells. Ultimately, after 96 h of incubation, the addition of 5 nM bortezomib to the combination of 10 μM rosiglitazone/25 μM bexarotene significantly enhances the antiproliferative activity in myeloma-derived ARH77 cells that showed resistance to single agent bortezomib (39% ±3.8 vs 73% ±1.2, P<0.05).
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O'Connor, Owen A., Bor-Sheng Ko, Ming-Chung Wang, Dai Maruyama, Yuqin Song, Ee-Min Yeoh, and Kensei Tobinai. "A Meta-Analysis of Pivotal Pralatrexate Studies in Relapsed/Refractory Mature T-Cell Lymphoma (r/r TCL)." Blood 138, Supplement 1 (November 5, 2021): 2450. http://dx.doi.org/10.1182/blood-2021-145580.
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Abstract Introduction While patients with r/r TCL have a poor prognosis, significant progress has been made over the past decade in developing new drugs and regimens for the disease. All of the approved agents have their own distinct advantages and issues, making it essential to critically assess the merits and limitations of each. It is well accepted that the development of acquired drug resistance to conventional chemotherapy reduces efficacy with each subsequent line of therapy. However in the era of novel single agents, less is known about the role of these new drugs in patients with chemo-sensitive and chemo-refractory disease. Based on results of the PROPEL trial in 111 treated patients, pralatrexate was the first drug approved as a novel single agent in the US for r/r TCL (O'Connor et al. J Clin Oncol. 2011). Drug registration and approval of pralatrexate was subsequently attained in many healthcare ministries around the world, including Japan, China and Taiwan based on the results of PROPEL and/or locally conducted studies (Maruyama et al. Cancer Sci 2017; Hong et al. Target Oncol 2019; Wang et al. EHA 2019). A meta-analysis of these four trials was undertaken to better understand those factors influencing efficacy and safety of pralatrexate in this patient population. Methods Patient- or summary-level data presented in the individual clinical study reports from the four regulatory-mandated prospective clinical trials of pralatrexate monotherapy in patients with r/r TCL were included in this meta-analysis. Pooled studies (phase 2 PDX-008 [PROPEL], phase 3 FOT12-CN-301, phase 4 FOT14-TW-401, phase 2 part of PDX-JP1) collected data on the primary efficacy endpoint of objective response rate (ORR) as assessed by central review. Secondary efficacy endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Data were collected in accordance with the original protocol and updated with longer survival follow-up where available. Relapse was defined as achieving a complete response (CR) or partial response (PR) on prior therapy lasting for > 3 months, and refractory was defined as stable disease or progressive disease on prior therapy or relapsed disease <3 months of achieving CR or PR. Both the fixed effect model (includes within study variations) and random effects model (includes within study and between study variations) were considered in this meta-analysis. Results A total of 221 patients were included in the efficacy analysis set for this meta-analysis. The median age was 59 years (range 21-89) and 67% were male. Histological subtypes as assessed by the central review included PTCL-not otherwise specified (108 pts, 49%), angioimmunoblastic T-cell lymphoma (47 pts, 21%), anaplastic large cell lymphoma (ALK-negative; 26 pts, 12%), transformed mycosis fungoides (12 pts, 5%), extranodal NK/T cell lymphoma, nasal type (7 pts, 3%), and other/missing (10/11 pts, 5% each). Patients had a median of 2 prior lines of systemic therapies (range 1-14). At the most recent line of therapy prior to pralatrexate, 68 (31%) and 103 (47%) patients were classified as relapsed and refractory respectively, with the remaining 50 (23%) unable to be determined due to response to the prior therapy being unavailable or not evaluable. Ten percent (n=24) of patients had stem cell transplant prior to receiving pralatrexate. Pooled ORR of the entire cohort was 40.7% (95% CI, 34.2, 47.5), including 30 in CR (13.6%) and 60 in PR (27.2%). The median DOR was 9.1 months (95% CI, 7.4-10.8). Median PFS was 4.6 months (95% CI, 4.0-5.2) while median OS was 16.3 months (95% CI, 13.1-22.6). Conclusions This meta-analysis confirms significant clinical activity of pralatrexate in heavily treated patients with r/r TCL, with an ORR of 40.7% at a median of two lines of prior therapy. These findings meaningfully bolster the original observations from PROPEL with 3 additional regulatory mandated clinical studies and suggest that earlier use may be associated with improved outcome. Further details on the efficacy by prior lines of systemic therapy as well as safety data will be presented. Disclosures O'Connor: TG Therapeutics: Current Employment, Current equity holder in publicly-traded company; Merck: Research Funding; BMS: Research Funding; Astex: Research Funding; Kymera: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; Mundipharma: Honoraria; Myeloid Therapeutics: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Maruyama: Novartis: Research Funding; Chugai: Honoraria, Research Funding; Amgen: Research Funding; BMS: Honoraria, Research Funding; Astellas Pharma,: Research Funding; Otsuka: Research Funding; MSD: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Ono: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria; Kyowa Kirin: Honoraria; Zenyaku: Honoraria; AstraZeneca: Honoraria; Nippon: Honoraria; SymBio: Honoraria. Yeoh: Mundipharma: Current Employment. Tobinai: Celgene: Consultancy, Honoraria; Chugai Pharmaceutical: Honoraria; Eisai: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; HUYA Bioscience International: Consultancy, Honoraria; Kyowa Kirin: Honoraria; Mundipharma: Consultancy, Honoraria; Ono Pharmaceutical: Consultancy, Honoraria; Solasia Pharma: Honoraria; Takeda Pharmaceutical: Consultancy, Honoraria; Yakult: Honoraria; Zenyaku Kogyo: Consultancy, Honoraria.
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McGregor, Stephanie, Anant Shah, Gordana Raca, Kamran Mirza, John Anastasi, James W. Vardiman, Elizabeth Hyjek, and Sandeep Gurbuxani. "PLZF Staining Identifies Peripheral T-Cell Lymphomas Derived From Non-Conventional T-Cells With Limited α-Chain Diversity." Blood 122, no. 21 (November 15, 2013): 4300. http://dx.doi.org/10.1182/blood.v122.21.4300.4300.
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Abstract Peripheral T-cell lymphomas are uncommon and account for 10-15% of all non-Hodgkin lymphomas (NHL). The current classification and treatment strategy of peripheral T-cell lymphomas relies on integrating morphology with immunophenotype, genetics and clinical presentation. However, the most common category of peripheral T-cell lymphomas remains peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) reflecting the lack of specific parameters to better define these lymphomas in a biologically relevant way. As our understanding of the biology of peripheral T-cell development continues to improve, several immunophenotypic markers have become available that can delineate peripheral T-cells into functional subsets. It is now recognized that peripheral T-cell lymphomas can arise from both conventional and innate-like T-cells. Classically, peripheral lymphomas with the γδ T-cell receptor (TCR) as well as lymphomas derived from true natural killer (NK) cells are considered to be arising from the innate T-cells whereas T-cell lymphomas with an αβ TCR are assumed to be derived from the adaptive, conventional T-cells. However, several recent studies have identified relatively rare populations of αβ T cells with extremely limited α chain diversity. These T-cells are characterized by the ability to mount immune responses by interacting with non-classical MHC class I antigen presenting molecules even in the absence of intentional priming. The cells within these populations express markers characteristic of NK cells and/or memory T-cells and include cells frequently labeled as “NKT” cells and mucosal-associated invariant T-cells (MAIT). Whether these cells contribute to peripheral T-cell lymphomas is not known. The transcription factor promyelocytic leukemia zinc finger (PLZF) is indispensable for development and maturation of these T-cells. We therefore asked the question whether PLZF expression could be used to identify peripheral T-cell lymphomas derived from these innate like non-conventional T-cells. To answer this question, we generated a tissue microarray that included biopsies from 26 PTCL-NOS, 11 anaplastic large cell lymphomas (ALCL), ALK-, and 13 ALCL, ALK+. Histologically normal tonsil, lymph node, thymus and gastrointestinal biopsies were used as controls. Immunohistochemistry with the PLZF antibody was performed in the clinical immunohistochemistry laboratory. Only rare PLZF positive cells were observed in uninvolved tonsils, lymph nodes, and thymus. In contrast, the intestinal mucosa, which is normally enriched in PLZF positive innate type T-cells showed a relative abundance with expression observed in 8-10% lymphocytes. Lymphomas were scored as positive when 20% or more of tumor cells showed expression with nuclear localization. Within the lymphomas, PLZF expression was observed in 2/26 PTCL-NOS and 2/13 ALCL, ALK+. PLZF expression was not observed in any of the ALCL, ALK- lymphomas included in the current study. PCR amplification followed by sequencing identified the Vα7.2-Jα33 TCR rearrangement characteristic of the MAIT cells in the two PLZF positive PTCL-NOS lymphomas confirming the origin of these lymphomas from bona fide innate-like T-cells. Sequencing of the TCR in the remaining three PLZF positive lymphomas is currently in progress. Cytogenetic analysis was available in three of the 4 cases. While t(2;5) was the sole cytogenetic abnormality in one ALCL, ALK+ lymphoma, the remaining two cases, including one ALCL, ALK+ had a complex karyotype that included t(2;5). In view of the relatively small number of patients available for analysis and the heterogeneous therapy administered to patients included retrospectively in the study, an outcome analysis was not performed. In conclusion, we demonstrate that PLZF expression identifies lymphomas derived from non-conventional innate-like T-cells and likely represent a biologically unique group of peripheral T-cell lymphoma. It is well known that innate T-cells are highly resistant to xenobiotics due to high expression of the transporter ATP binding cassette B1 (MDR). Prospective evaluation for PLZF expression may therefore be useful in identifying patients who will benefit from therapy that specifically targets this pathway of drug resistance. Disclosures: No relevant conflicts of interest to declare.