Статті в журналах з теми "BRAF isoforms"

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS.

Cutaneous melanoma is the most aggressive form of skin cancer. Despite the significant advances in the management of melanoma in recent decades, it still represents a challenge for clinicians. The TP53 gene, the guardian of the genome, which is altered in more than 50% of human cancers, is rarely mutated in melanoma. More recently, researchers started to appreciate the importance of shorter p53 isoforms as potential modifiers of the p53-dependent responses. We analyzed the expression of p53 and p73 isoforms both at the RNA and protein level in a panel of melanoma-derived cell lines with different TP53 and BRAF status, in normal conditions or upon treatment with common anti-cancer DNA damaging agents or targeted therapy. Using lentiviral vectors, we also generated stable clones of H1299 p53 null cells over-expressing the less characterized isoforms Δ160p53α, Δ160p53β, and Δ160p53γ. Further, we obtained two melanoma-derived cell lines resistant to BRAF inhibitor vemurafenib. We observed that melanoma cell lines expressed a wide array of p53 and p73 isoforms, with Δ160p53α as the most variable one. We demonstrated for the first time that Δ160p53α, and to a lesser extent Δ160p53β, can be recruited on chromatin, and that Δ160p53γ can localize in perinuclear foci; moreover, all Δ160p53 isoforms can stimulate proliferation and in vitro migration. Lastly, vemurafenib-resistant melanoma cells showed an altered expression of p53 and p73 isoforms, namely an increased expression of potentially pro-oncogenic Δ40p53β and a decrease in tumor-suppressive TAp73β. We therefore propose that p53 family isoforms can play a role in melanoma cells’ aggressiveness.

Metastatic melanoma is one of the most aggressive tumors, with frequent mutations affecting components of the MAPK pathway, mainly protein kinase BRAF. Despite promising initial response to BRAF inhibitors, melanoma progresses due to development of resistance. In addition to frequent reactivation of MAPK or activation of PI3K/AKT signaling pathways, recently, the p53 pathway has been shown to contribute to acquired resistance to targeted MAPK inhibitor therapy. Canonical tumor suppressor p53 is inactivated in melanoma by diverse mechanisms. The TP53 gene and two other family members, TP63 and TP73, encode numerous protein isoforms that exhibit diverse functions during tumorigenesis. The p53 family isoforms can be produced by usage of alternative promoters and/or splicing on the C- and N-terminus. Various p53 family isoforms are expressed in melanoma cell lines and tumor samples, and several of them have already shown to have specific functions in melanoma, affecting proliferation, survival, metastatic potential, invasion, migration, and response to therapy. Of special interest are p53 family isoforms with increased expression and direct involvement in acquired resistance to MAPK inhibitors in melanoma cells, implying that modulating their expression or targeting their functional pathways could be a potential therapeutic strategy to overcome resistance to MAPK inhibitors in melanoma.

The mitogen-activated protein kinase (MAPK) pathway, consisting of the Ras-Raf-MEK-ERK signaling cascade, regulates genes that control cellular development, differentiation, proliferation, and apoptosis. Within the cascade, multiple isoforms of Ras and Raf each display differences in functionality, efficiency, and, critically, oncogenic potential. According to the NCI, over 30% of all human cancers are driven by Ras genes. This dysfunctional signaling is implicated in a wide variety of leukemias and solid tumors, both with and without viral etiology. Due to the strong evidence of Ras-Raf involvement in tumorigenesis, many have attempted to target the cascade to treat these malignancies. Decades of unsuccessful experimentation had deemed Ras undruggable, but recently, the approval of Sotorasib as the first ever KRas inhibitor represents a monumental breakthrough. This advancement is not without novel challenges. As a G12C mutant-specific drug, it also represents the issue of drug target specificity within Ras pathway; not only do many drugs only affect single mutational profiles, with few pan-inhibitor exceptions, tumor genetic heterogeneity may give rise to drug-resistant profiles. Furthermore, significant challenges in targeting downstream Raf, especially the BRaf isoform, lie in the paradoxical activation of wild-type BRaf by BRaf mutant inhibitors. This literature review will delineate the mechanisms of Ras signaling in the MAPK pathway and its possible oncogenic mutations, illustrate how specific mutations affect the pathogenesis of specific cancers, and compare available and in-development treatments targeting the Ras pathway.

616 Background: BRAF V600E mutations, present in <10% of patients with metastatic colorectal cancer (mCRC), are associated with low responses to chemotherapy and poor survival outcomes. Targeted therapies against BRAF and EGFR have shown promising clinical activity. The panRAF inhibitor (PRI) LSN3074753 inhibits dimerization of all RAF isoforms to impede downstream MEK activation, with no reflexive MAPK reactivation common with other BRAF inhibitors. Anti-tumor activity of PRI has not been compared to BRAF + EGFR inhibition in patient-derived xenograft (PDX) models of BRAF V600E mCRC. Methods: Two PDX models of BRAF V600E mCRC (B1003 and C0999) were generated. C0999 featured a concomitant KRAS G12D mutation following resistance to the BRAF V600E kinase inhibitor vemurafenib. Mice were treated daily with oral PRI or with the combination of vemurafenib + intraperitoneal cetuximab. Tumor volumes were measured twice weekly. B1003 and C0999 cell cultures were established to test the interaction between PRI and palbociclib or BYL319 (PI3K inhibitor). Results: PRI was tolerated at a dose of 60 mg/kg and demonstrated a reduced tumor volume in the B1003 model after 28 days when compared to untreated controls (P=.03). No difference in tumor volume was seen between PRI and vemurafenib + cetuximab (P=.08). Assessment of anti-tumor activity by PRI in the vemurafenib-resistant BRAF V600E/KRAS G12D C0999 model will be reported. Cell culture from both the B1003 and C0999 models demonstrated synergism for PRI with palbociclib (ED50 .41 and .62 for the 2 models, respectively) and with BYL319 (ED50 .48 and .86, respectively). Conclusions: panRAF inhibition demonstrates similar anti-tumor activity to BRAF + EGFR inhibition in PDX models of BRAF V600E and represents a promising treatment strategy for further combination studies targeting additional critical signaling pathways in mCRC.

Cutaneous melanoma (CM) is the most lethal tumor among skin cancers, and its incidence is constantly increasing. A deeper understanding of the molecular processes guiding melanoma pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs play a key role in melanoma biology. Recently, next generation sequencing (NGS) experiments, designed to assess small-RNA expression, revealed the existence of microRNA variants with different length and sequence. These microRNA isoforms are known as isomiRs and provide an additional layer to the complex non-coding RNA world. Here, we collected data from NGS experiments to provide a comprehensive characterization of miRNA and isomiR dysregulation in benign nevi (BN) and early-stage melanomas. We observed that melanoma and BN express different and specific isomiRs and have a different isomiR abundance distribution. Moreover, isomiRs from the same microRNA can have opposite expression trends between groups. Using The Cancer Genome Atlas (TCGA) dataset of skin cancers, we analyzed isomiR expression in primary melanoma and melanoma metastasis and tested their association with NF1, BRAF and NRAS mutations. IsomiRs differentially expressed were identified and catalogued with reference to the canonical form. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import–export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Abstract The PI3K/AKT pathway is frequently dysregulated in cutaneous melanoma and impacts both tumor aggression and resistance to BRAFV600E/K inhibitors. While current clinical approaches to AKT inhibition are severely limited by the toxicity of pan-AKT inhibitors, selective inhibition of individual AKT isoforms (AKT1, AKT2, or AKT3) remains an attractive, if yet unattainable, approach. A critical gap in our understanding concerns how the three highly homologous yet functionally distinct AKT isoforms contribute to melanomagenesis and treatment response. To address this question, we are employing RNAi and gene editing approaches to interrogate the effect of selective suppression or CRISPR/Cas9-mediated deletion of each isoform in vitro, as well as the impact of AKT isoform loss on 1) the growth of melanoma xenografts and 2) the development of spontaneous tumors in a melanoma-prone mouse model. In addition, we are interrogating tumor-promoting functions of AKT isoform-selective substrates recently identified in a phospho-proteomic screen of mouse fibroblasts, a system that allows us to identify downstream actionable targets that may mediate the effects of AKT isoforms in tumorigenesis. Broadly, we are focusing on the importance of AKTs in tumor cell growth, metastasis, and response to inhibitors of BRAFV600E/K or CDK4/6. We find that loss of AKT1 impacts growth of BRAF mutant human melanoma cells both in vitro and in vivo. Additionally, AKT1 appears to play an isoform-specific role in response to pharmacologic CDK4/6 inhibition, impacting transcriptional and morphologic changes typically associated with permanent cell cycle arrest. In contrast, loss of AKT2 has a minimal impact on melanoma cell growth or response to CDK4/6 inhibition, but severely limits the development of metastatic disease, potentially through a combination of impaired seeding at the metastatic site and defects in glycolysis. While AKT3 loss does not appreciably impact the above-mentioned cellular phenotypes, the protumorigenic role of AKT3 may involve activation of broadly-acting neutral proteases previously implicated in cell cycle progression, cell migration, and CDK5 regulation. Taken together, we provide evidence for distinct roles for AKT isoforms in several aspects of the tumorigenic process as well as response to current therapies. Future studies will focus on identification and targeting of the relevant downstream mediators of these phenotypes. This abstract is also being presented as Poster A16. Citation Format: Jaymes Farrell, Jodie Pietruska, Siobhan McRee, Philip Tsichlis, Philip Hinds. Defining isoform-specific roles for AKTs in BRAFV600E-driven melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR14.

Medulloblastomas and ependymomas are the most common malignant brain tumors in children. However genetic abnormalities associated with their development and prognosis remain unclear. Recently two gene fusions, KIAA1549–BRAF and SRGAP3–RAF1 have been detected in a number of brain tumours. We report here our development and validation of RT-RQPCR assays to detect various isoforms of these two fusion genes in formalin fixed paraffin embedded (FFPE) tissues of medulloblastoma and ependymoma. We examined these fusion genes in 44 paediatric brain tumours, 33 medulloblastomas and 11 ependymomas. We detected both fusion transcripts in 8/33, 5/33 SRGAP3 ex10/RAF1 ex10, and 3/33 KIAA1549 ex16/BRAF ex9, meduloblastomas but none in the 11 ependymomas examined. This investigation provided evidence to the value of RT-RQPCR assays for the detection of these fusion genes in large-scale studies on FFPE tissues. The study also reports the first detection of RAF fusion genes in meduloblstomas.

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Targeted therapy is increasingly used to manage metastatic papillary thyroid cancer. The focus of the present study was to examine glucose metabolism and tumor responses for thyroid cancer xenografts expressing the glycolytic pathway modulators platelet-derived growth factor receptor (PDGFR) and BRAFV600E. Radiolabelled glucose derivative [18F]FDG was used to analyze the effects of PDGFR blockade with imatinib, BRAF blockade with vemurafenib, as well as combined PDGFR and BRAF blockade in vitro and in vivo with PET. Dynamic PET data was correlated with immunohistochemistry staining and kinetic analysis for facilitative glucose transporter 1 (GLUT1) and hexokinase-II (HK2). Vemurafenib decreased [18F]FDG uptake in BCPAP cells in vitro; however, it was increased by ~70% with imatinib application to BCPAP cells. This metabolic response to tyrosine kinase inhibition required BRAFV600E as it was not seen in cell lines lacking mutated BRAF (TPC1). In xenografts, imatinib therapy in BCPAP thyroid tumour-bearing mice significantly increased [18F]FDG uptake and retention (>30%) in BCPAP tumours with PDGFRβ or both (α+β) isoforms. Kinetic analysis revealed that the increased glucose uptake is a consequence of increased phosphorylation and intracellular trapping of [18F]FDG confirmed by an increase in HK2 protein expression and activity, but not GLUT1 activity. BRAF inhibition alone, or combined PDGFR and BRAF inhibition, reduced (~60%) [18F]FDG uptake in both types of BCPAP (β or α+β) tumours. In terms of tumour growth, combination therapy with imatinib and vemurafenib led to a near abolition of the tumors (~90% reduction), but single therapy for BCPAP with PDGFRα expression was much less effective. In summary, imatinib led to a paradoxical increase of [18F]FDG uptake in xenografts that was reversed through BRAFV600E inhibition. The present data show that metabolic reprogramming in thyroid cancer occurs as a consequence of BRAF-mediated upregulation of HK2 expression that may permit tumour growth with isolated blockade of upstream tyrosine kinase receptors.

More than 95% of the 20,000 to 25,000 transcribed human genes undergo alternative RNA splicing, which increases the diversity of the proteome. Isoforms derived from the same gene can have distinct and, in some cases, opposing functions. Accumulating evidence suggests that aberrant RNA splicing is a common and driving event in cancer development and progression. Moreover, aberrant splicing events conferring drug/therapy resistance in cancer is far more common than previously envisioned. In this review, aberrant splicing events in cancer-associated genes, namely BCL2L1, FAS, HRAS, CD44, Cyclin D1, CASP2, TMPRSS2-ERG, FGFR2, VEGF, AR and KLF6, will be discussed. Also highlighted are the functional consequences of aberrant splice variants (BCR-Abl35INS, BIM-γ, IK6, p61 BRAF V600E, CD19-∆2, AR-V7 and PIK3CD-S) in promoting resistance to cancer targeted therapy or immunotherapy. To overcome drug resistance, we discuss opportunities for developing novel strategies to specifically target the aberrant splice variants or splicing machinery that generates the splice variants. Therapeutic approaches include the development of splice variant-specific siRNAs, splice switching antisense oligonucleotides, and small molecule inhibitors targeting splicing factors, splicing factor kinases or the aberrant oncogenic protein isoforms.

Abstract Abstract 3392 Introduction: Chronic myelogenous leukemia (CML) is characterized by the t(9;22)(q34;q11) chromosomal translocation and the expression of BCR-ABL, a fusion protein with tyrosine kinase activity. BCR-ABL activates various signaling cascades mediating signals for proliferation, transformation and anti-apoptosis. The BCR-ABL inhibitor imatinib is the standard therapy for CML. However, this treatment is assumed to be not curative since leukemia initiating cells cannot be completely eradicated by solely BCR-ABL inhibition. Identification of key mediators within the BCR-ABL signaling cascade thus remains crucial. The MEK/ERK cascade is one of the major promitogenic pathways activated in CML. Whether Raf-1, BRAF or both Raf isoforms are required for BCR-ABL mediated activation of this pathway is not known. As both Raf-1 and BRAF knockout mice are embryonic lethal, the role of Raf-1 and BRAF in BCR-ABL mediated leukemogenesis has not been investigated in appropriate in vivo models so far. Here we studied the impact of Raf-1 and BRAF for BCR-ABL dependent transformation by using a retroviral vector system, which allows to directly couple shRNA based target suppression to oncogene expression in a CML mouse model. Methods: We exerted an shRNA-based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the BCR-ABL oncogene and miR-30 based shRNAs (miR) for BRAF and Raf-1 respectively on a single construct resulting in one shared RNA transcript. This approach ensured knockdowns of more than 80–90% for the respective Raf protein in every BCR-ABL transformed cell. Result: Methylcellulose assays showed that primary bone marrow cells coexpressing Raf-1 miR and BCR-ABL had a 2 fold decreased colony forming ability, whereas BRAF knockdown had no impact on colony forming ability compared to control cells. We then transplanted murine bone marrow (BM), transduced with retrovirus coexpressing Raf-1 or BRAF miR and p185 BCR-ABL, to lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf-1 miR but not BRAF miR and BCR-ABL compared with the BCR-ABL transduced control miR group. Raf-1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival in comparison to control mice. However, BRAF knockdown had no significant effect on overall survival or disease progression in the bone marrow transduction transplantation model. We hypothesized that this impact of Raf-1 knockdown might be due to incomplete activation of the MEK/ERK cascade in the absence of Raf-1. We could demonstrate that Raf-1 is necessary for BCR-ABL dependent ERK activation in primary murine bone marrow as well as in cell lines. In contrast in BRAF knockdown BCR-ABL positive cells levels of phosphorylated and thereby activated ERK remained unchanged compared to control cells, indicating that BRAF is dispensable for BCR-ABL dependent ERK phosphorylation. Conclusion: Taken together our data demonstrate that primarily Raf-1 is responsible for BCR-ABL mediated activation of the promitogenic MEK/ERK signaling cascade. Raf-1 but not BRAF is also crucial for the development of a myeloproliferative disease by BCR-ABL in mice. Therefore, Raf-1 but not BRAF inhibition may be a potential interesting additional therapeutic approach in CML.The coexpression of an oncogene and a target specific miR-30 based shRNA from a single retroviral construct displays a powerful tool that can be used to systematically screen drugable signaling targets involved in CML and other leukemic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract FDA approved three RAF inhibitors for the treatment of tumors containing BRAFV600 mutations, but one of the major drawbacks of these type I RAF inhibitors is to activate MAPK signaling pathway, instead of inhibiting signaling, which is referred to as paradoxical activation. Such undesired paradoxical activation not only leads to renewed tumor growth but also spurs additional cancer growth in non-cancerous wild-type BRAF tissue. Plus, these first-generation RAF inhibitors targeting BRAFV600 mutants are unable to inhibit oncogenic RAF dimers. This has led to the development of type II RAF inhibitors such as belvarafenib and day101 to block the activity of multiple forms of RAF while avoiding paradoxical activation. Two lead-like stage compounds 1 and 2 were specifically designed as type II RAF inhibitors to have activity across RAF isoforms including BRAFV600E, BRAF wild-type, and CRAF, showing higher potency than competitors. Compounds 1 and 2 potently inhibited the growth of BRAFV600E melanoma cells and NRAS or KRAS mutant cancer cells. Compounds 1 and 2 promoted the formation of BRAF/CRAF heterodimers by directly binding to the RAF kinase domain like other type II RAF inhibitors and inhibited phosphorylation of downstream effectors MEK and ERK in a dose-dependent manner in RAS mutant cancer cells, suggesting less paradoxical activation liability. Compound 2 showed superior on-target inhibitory activity for BRAFV600E, BRAF wild-type, and CRAF than belvarafenib through RAF immunoprecipitation (IP) kinase assay. Inhibition of the RAF downstream signaling was also confirmed by quantifying the level of phospho-ERK in NRAS or KRAS mutant cancer cells. In HCT116 (KRASG13D) subcutaneous xenograft model, compound 1 showed tumor growth inhibition efficacy, suggesting a potential to address RAS mutant driven- as well as BRAFV600 mutant driven tumors. Also, combination treatment with MEK inhibitor and/or immune checkpoint inhibitor would further improve the therapeutic activity and expand target indication for unmet medical needs. Citation Format: Kwangwoo Hwang, SeoHyun Jo, Jieun Choi, Ga-young Choi, Jiseon Choi, Ji-Hye Kwon, Dong-Guk Shin, Jiyeon Kim, Se-Hyuk Kim, Haelee Kim, Ha Yeon Cho, Jung Beom Son, Nam Doo Kim, Hwan Geun Choi, Daekwon Kim, Sunghwan Kim. Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB521.

Abstract Background Erdheim–Chester disease (ECD) is a rare histiocytic neoplasm characterized by recurrent alterations in the MAPK (mitogen-activating protein kinase) pathway. The existing literature about the neuro-oncological spectrum of ECD is limited. Methods We present retrospective clinical, radiographic, pathologic, molecular, and treatment data from 30 patients with ECD neurohistiocytic involvement treated at a tertiary center. Results Median age was 52 years (range, 7–77), and 20 (67%) patients were male. Presenting symptoms included ataxia in 19 patients (63%), dysarthria in 14 (47%), diabetes insipidus in 12 (40%), cognitive impairment in 10 (33%), and bulbar affect in 9 (30%). Neurosurgical biopsy specimens in 8 patients demonstrated varied morphologic findings often uncharacteristic of typical ECD lesions. Molecular analysis revealed mutations in BRAF (18 patients), MAP2K1 (5), RAS isoforms (2), and 2 fusions involving BRAF and ALK. Conventional therapies (corticosteroids, immunosuppressants, interferon-alpha [IFN-α], cytotoxic chemotherapy) led to partial radiographic response in 8/40 patients (20%) by MRI with no complete responses, partial metabolic response in 4/16 (25%), and complete metabolic response in 1/16 (6%) by 18F-fluorodeoxyglucose (FDG)-PET scan. In comparison, targeted (kinase inhibitor) therapies yielded partial radiographic response in 10/27 (37%) and complete radiographic response in 14/27 (52%) by MRI, and partial metabolic response in 6/25 (24%) and complete metabolic response in 17/25 (68%) by FDG-PET scan. Conclusions These data highlight underrecognized symptomatology, heterogeneous neuropathology, and robust responses to targeted therapies across the mutational spectrum in ECD patients with neurological involvement, particularly when conventional therapies have failed.

4064 Background: DUSPs dephosphorylate P-MAPK and are activated as a negative feedback loop upon RTK signaling. Higher expression of DUSP 4 & 6 is also found in cells with constitutive Erk activation like KRAS mutant (MUT) cells (Bild et al. Nature 2005). We correlated DUSP1, 4, 6 (isoforms a & b) & 8 mRNA expression level in FFPE primary colorectal cancer (CRC) of 186 chemorefractory patients treated with cetuximab (CTX) with KRAS MUT state and progression-free survival (PFS) and overall survival (OS). Methods: KRAS codon 12,13, 61&146, BRAF V600E and NRAS codon 12&13 MUT were analyzed with the Sequenom MALDI TOF MassArray system. The DUSPs and 3 housekeeping genes were quantified by qRT-PCR. TwoStep cluster analysis was performed. PFS and OS were estimated by the Kaplan-Meier method. Results: KRAS MUT was associated with increased DUSP4 (MWU;p=.0006) & 6a (p=.0067). DUSP6a dephosphorylates P-Erk, DUSP4 also dephosphorylates P-JNK & P-p38. DUSP1 & 8 primarily dephosphorylate P-JNK & P-p38 and were not associated with KRAS MUT. KRAS MUT clustered into 3 groups according to DUSP4 expression: 32 high, 38 median & 13 low (t- test;p<.0001). The low MUT expression was comparable to wild-type (WT) expression. KRAS WT clustered into 2 groups: 69 low & 24 high DUSP4 (ANOVA;p<.0001). 7/24 of high expressors were found to have a BRAF or NRAS MUT. The 32 MUT high expressors had a longer median PFS (log-rank;p=.015) and OS (p=.065) after CTX. The 17 KRAS/BRAF/NRAS WT high expressors had a shorter median OS (p=.026), but not PFS (p=.745). Conclusions: There is a significantly higher DUSP4 & 6a mRNA expression in the KRAS MUT compared to WT primary CRC. However, this is not a black and white observation. In the KRAS MUT there are 3 distinct clusters of DUSP4 expression. The high expressors (= supposed attenuated Erk signaling) have a longer PFS and OS after CTX. Adaptation to constitutive KRAS signaling with differential levels of MEK/Erk activation needs to be further investigated and will be of help in selecting patients for therapy with MEK inhibitors. It suggests not all KRAS MUT will be good candidates for MEK inhibitors. In the cluster of high DUSP4 expressing KRAS WT 30% are BRAF or NRAS MUT. This suggests DUSP expression of FFPE samples could be a more sensitive marker of MEK/Erk activation and resistance to EGFR inhibitors than KRAS MUT analysis alone. [Table: see text]

Background and objectives: Cancer represents the miscommunication between and within the body cells. The mutations of the oncogenes encoding the MAPK pathways play an important role in the development of tumoral diseases. The mutations of KRAS and BRAF oncogenes are involved in colorectal cancer and melanoma, while the NRAS mutations are associated with melanoma. Thiazolidine-2,4-dione is a versatile scaffold in medicinal chemistry and a useful tool in the development of new antitumoral compounds. The aim of our study was to predict the pharmacokinetic/pharmacodynamic properties, the drug-likeness and lead-likeness of two series of synthetic 5-arylidene(chromenyl-methylene)-thiazolidinediones, the molecular docking on the oncoproteins K-Ras, N-Ras and B-Raf, and to investigate the cytotoxicity of the compounds, in order to select the best structural profile for potential anticancer agents. Materials and Methods: In our paper we studied the cytotoxicity of two series of thiazolidine-2,4-dione derivatives, their ADME-Tox properties and the molecular docking on a mutant protein of K-Ras, two isoforms of N-Ras and an isoform of B-Raf with 16 mutations. Results: The heterocyclic compounds strongly interact with K-Ras and N-Ras right after their posttranslational processing and/or compete with GDP for the nucleotide-binding site of the two GTPases. They are less active against the GDP-bound states of the two targets. All derivatives have a similar binding pattern in the active site of B-Raf. Conclusions: The data obtained encourage the further investigation of the 5-arylidene(chromenyl-methylene)-thiazolidinediones as potential new agents against the oncoproteins K-Ras, N-Ras and B-Raf.

Melanoma is the most aggressive type of skin cancer. Despite the available therapies, the minimum residual disease is still refractory. Reactive oxygen and nitrogen species (ROS and RNS) play a dual role in melanoma, where redox imbalance is involved from initiation to metastasis and resistance. Redox proteins modulate the disease by controlling ROS/RNS levels in immune response, proliferation, invasion, and relapse. Chemotherapeutics such as BRAF and MEK inhibitors promote oxidative stress, but high ROS/RNS amounts with a robust antioxidant system allow cells to be adaptive and cooperate to non-toxic levels. These proteins could act as biomarkers and possible targets. By understanding the complex mechanisms involved in adaptation and searching for new targets to make cells more susceptible to treatment, the disease might be overcome. Therefore, exploring the role of redox-sensitive proteins and the modulation of redox homeostasis may provide clues to new therapies. This study analyzes information obtained from a public cohort of melanoma patients about the expression of redox-generating and detoxifying proteins in melanoma during the disease stages, genetic alterations, and overall patient survival status. According to our analysis, 66% of the isoforms presented differential expression on melanoma progression: NOS2, SOD1, NOX4, PRX3, PXDN and GPX1 are increased during melanoma progression, while CAT, GPX3, TXNIP, and PRX2 are decreased. Besides, the stage of the disease could influence the result as well. The levels of PRX1, PRX5 and PRX6 can be increased or decreased depending on the stage. We showed that all analyzed isoforms presented some genetic alteration on the gene, most of them (78%) for increased mRNA expression. Interestingly, 34% of all melanoma patients showed genetic alterations on TRX1, most for decreased mRNA expression. Additionally, 15% of the isoforms showed a significant reduction in overall patient survival status for an altered group (PRX3, PRX5, TR2, and GR) and the unaltered group (NOX4). Although no such specific antioxidant therapy is approved for melanoma yet, inhibitors or mimetics of these redox-sensitive proteins have achieved very promising results. We foresee that forthcoming investigations on the modulation of these proteins will bring significant advances for cancer therapy.

Abstract Chronic lymphocytic leukemia (CLL) remains incurable with current state of the art therapy creating the need for novel therapeutic concepts. Kinase inhibitors represent a promising strategy in the treatment of various malignancies including CLL. However, based on the recent experience with other targeted therapy compounds used as single agents, it appears important to identify additional targets and to evaluate therapeutic combinations targeting two or more critical signaling hubs in CLL cells. This strategy is likely to counteract the development of drug resistance more efficiently. We and others recently showed that the Raf/MEK/ERK pathway plays a critical role for the in vitro survival of CLL cells and demonstrated that drugs such as sorafenib targeting all Raf-isoforms and other kinases, induce apoptosis. Here, we provide a detailed analysis of various B-Raf inhibitors, including sorafenib, PLX4720 (vemurafenib tool compound) and dabrafenib on apoptotic pathways in primary human CLL cells and their impact on CLL cell survival alone or in combination with MEK (U0126), dual phosphoinositide-3 kinase/mTOR inhibitor (BEZ235), and IGF1R inhibitors (AG1024, Picropodophyllin (PPP)). 10µM sorafenib, a concentration comparable to plasma levels of this drug in patients, strongly induced apoptosis in CLL cells, while the more specific B-Raf inhibitor PLX4720 did not affect viability. This discrepant finding may be attributed to PLX4720’s well-described paradoxical ERK activation reported in various solid tumor types lacking BRAF mutations and containing increased Ras-GTP levels. Indeed, only sorafenib reduced ERK phosphorylation in CLL cells, while PLX4720 treatment even enhanced ERK activation. Interestingly, not only the B-Raf inhibitors PLX4720 or dabrafenib, but also lower doses (1-5µM) of sorafenib induced paradoxical ERK activation. Most likely, at these concentrations drug-bound Raf molecules act as potent allosteric activators on drug-free Raf monomers, thereby leading to an increase in ERK pathway activity. Importantly, paradoxical ERK activation coincided with enhanced viability. In line with findings in melanoma cells, BRAF (V600E) mutated CLL cells showed no paradoxical ERK activation and were effectively killed by the respective compounds. To identify inhibitor combinations reducing the in vitro survival of BRAF wild-type CLL isolates, we combined the B-Raf inhibitors with the MEK inhibitor U0126 or the dual PI3K/mTOR inhibitor BEZ235 observing reduced ERK phosphorylation and significantly enhanced rates of cell death (p<0.0009 for U0126; p<0.01 for BEZ235). This indicates that the additional MEK or PI3K inhibition counteracts paradoxical ERK activation in vitro and may overcome the resistance to apoptosis induction mediated by paradoxical ERK activation. Likewise, we tested combinatory effects of IGF1R inhibitors (AG1024, PPP) with suboptimal sorafenib doses and observed a significantly enhanced cell death for the combinations AG1024 and sorafenib (p<0,0001) as well as PPP and sorafenib (p<0,0001). In conclusion, this is the first description of a paradoxical ERK activation by Raf inhibitors in CLL cells with unmutated BRAF. Our observation of paradoxical ERK activation after treatment with suboptimal sorafenib doses may be of clinical importance since sorafenib is currently investigated in clinical trials in several malignancies including CLL. Our findings imply that Sorafenib plasma concentrations should be monitored when used for the treatment of CLL since lower plasma levels might promote paradoxical ERK activation and CLL progression. Furthermore, our results show the potential of therapies combining kinase inhibitors: additional MEK or PI3K inhibition or the concomitant inhibition of both pathways by IGF1R inhibitors may overcome paradoxical ERK activation. Furthermore, given our recent observation that the IGF1R is overexpressed in CLL cells, its inhibition by IGF1R kinase inhibitors might be advantageous over the single inhibition of MAPK or PI3K pathway components. Disclosures: No relevant conflicts of interest to declare.

T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAFV600E-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.

Background. The heat shock protein 90 (HSP90s) family is composed of molecular chaperones composed of four isoforms in humans, which has been widely reported as unregulated in various kinds of cancers. Nevertheless, the role of each HSP90s isoform in prognosis and immune infiltration in distinct subtypes of breast cancer (BRAC) remains unclear. Methods. Public online databases including the Oncomine, UALCAN, Kaplan-Meier Plotter, Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), GeneMANIA, and Database for Annotation, Visualization, and Integrated Discovery (DAVID) were integrated to perform bioinformatic analyses and to explore the possible associations among HSP90s gene expression, prognosis, and immune infiltration in BRAC. Results. The mRNA expression of all HSP90s members was elevated in distinct clinical stages and subtypes of BRAC, compared with the normal breast tissue ( P < 0.05 ). Overexpressed HSP90AA1 was associated with poor prognosis, particularly, both short overall survival (OS) and release-free survival (RFS) in Basal-like BRAC patients; overexpressed HSP90AB1 and HSP90B1 were both associated with poor RFS in Luminal A BRAC patients, while overexpressed TRAP1 was associated with favorable RFS in Luminal A BRAC patients. Moreover, HSP90s gene expression in BRAC showed correlations with the infiltration of CD8+ T cells, neutrophils, macrophages, and dendritic cells (DCs), as well as the activation of tumor-associated macrophages (TAMs), DCs, and CD4+ helper T (Th) cells. The underlying mechanisms of HSP90s modulating tumor-infiltrating immune cells (TIICs) might be related with their functions in antigen processing and presentation, major histocompatibility complex (MHC) binding, and assisting client proteins. Conclusion. This study demonstrated that HSP90s family genes were overexpressed and might be serve as prognostic biomarkers in subtypes of BRAC. It might be a novel breakthrough point of BRAC treatment to regulate immune infiltration in BRAC microenvironment for more effective anticancer immunity through pharmacological intervention of HSP90s.

Contemporary discoveries of fundamental science in recent decades in the field of oncology have led to the emergence of new highly effective anticancer drugs: targeted drugs and immune checkpoint inhibitors, use of which has made a breakthrough in the treatment of oncological diseases, including skin melanoma. Melanoma is still one of the most cancerous tumors. The number of patients resistant to targeted therapy and immunotherapy increases in the world every year. Oncologists have practically no leverage to influence the disease after the development of resistance to this type of therapy. In this regard, scientists around the world are looking for new application points for targeted drugs. Nowadays, the most common treatment method is BRAF inhibitors, since the BRAF mutation is detected in 40–60 % of patients with skin melanoma. However, the resistance to BRAF inhibitor therapy occur in half cases after 6–8 months. To overcome the resistance to the target therapy is one the most important issue, the studying of new isoform of anaplastic lymphoma kinase (ALK) may help to solve this problem.Purpose of the study – to order the data of the leading researchers of a new isoform of ALK, and reveal the most promising directions for its further progress.In the article, there are comparisons and analyses the 6 of the largest studies over the past 5 years devoted to a new isoform of ALK.The joint inhibition of the new ALK isoform and BRAFV600 showed positive results in several studies with different levels of ALKATI expression (alternative initiation of ALK transcription). The new ALK isoform can stimulate oncogenesis only within a certain “threshold” level of expression. Immunohistochemical examination cannot be the main method for determining the expression of a new ALK isoform due to low sensitivity. In almost all studies, tumors with ALK translocation responded to therapy with ALK inhibitors.Even though that the role of the new ALK isoform has been studied in recent years, the optimal method for evaluating the expression of ALKATI in routine practice has not yet been determined. Additional studies are also needed to understand the effectiveness of the use of ALК inhibitors in combination with BRAF and ERK inhibitors. Of interest is the blockade of extracellular vesicles and the study of the role of interleukin-3 in the inhibition of ALKATI.

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

Abstract The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT0145429) is a longitudinal study of 1000 patients with newly-diagnosed multiple myeloma. The study opened July 2011 and now includes over 650 patients from 91 sites in the United States, Canada and European Union. Each patient is required to receive an approved proteasome inhibitor, immunumodulatory agent, or both. Enriched tumor and matched constitutional samples are comprehensively analyzed using Long-Insert Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES) and RNA sequencing (RNAseq). Clinical parameters, Quality of Life measurements and health care resource utilization values are collected at study entry and every three months for a minimum of five years. Additional bone marrow aspirates are collected and analyzed at each recurrence or progression of disease. An extensive clinical and molecular database, the MMRF Researcher Gateway (https://research.themmrf.org), has been developed to facilitate the rapid dissemination of the results and provides the myeloma community with a mechanism to analyze the data. In this current interim analysis, we report on 195 patients that are fully characterized at the molecular level. We focused this analysis on immunoglobulin translocations and inter-chromosomal fusion transcripts. As expected we detected the classic canonical t(4;14), t(6;14), t(11;14), and t(14;16) translocations targeting FGFR3/MMSET, CCND3, CCND1, and MAF respectively. Seven patients presented with t(8;14) rearrangements correlating with high expression of MYC. Novel translocations were detected targeting MAP3K14/NIK in two patients and NFKB1, TOP1MT, TXNDC2, APOL3, FCHSD2, PRICKLE1, and BCL2L1 in individual patients. Importantly, the matched RNAseq data confirmed the high expression of MAP3K14, NFKB1, TOP1MT, APOL3 and BCL2L1. Moreover, the anti-apoptotic isoform of BCL2L1, Bcl-xL, was the prominent transcript isoform detected. In several patients we detected multiple IgH translocations. For instance the BCL2L1 translocation occurred in a downstream class switch recombination region from one associated with a co-occurring t(11:14). We also analyzed the RNAseq dataset for inter-chromosomal fusion transcripts and leveraged the independent long-insert WGS data to validate the predicted fusions. The only recurrent fusion partner identified was IgH-MMSET created by t(4:14). Fusion transcripts were detected in individual patients between IgH elements and MYEOV and WWOX along with several of the novel IgH translocation partners; NFKB1, TOP1MT, and APOL3. Several genes are involved in multiple fusions but with different partners. Three independent fusions were detected between the highly expressed gene FCHSD2 and MYC, MAP3K14, and ANKRD55. Three additional fusions were detected between MAP3K14 and ELL, PLCG2, and CDC27, which produce hybrid MAP3K14 isoforms lacking the N-terminal negative regulatory domain. We also detected three independent fusions involving BRF1, which is typically not expressed in myeloma tumors. These appear to be markers of translocations occurring just centromeric of the strong 3’ IgH enhancers. Interestingly, two of the partners are located in a region of chromosome 12 harboring MDM2 and spiked expression of MDM2 was observed. Additional genes with multiple fusion events included NEDD9 and ARHGEF12. Integrating the WES and RNAseq datasets, we identified 3518 variants (median 14 per patient) where the variant allele detected by WES, was also detected in the RNAseq data, suggesting it is potentially biologically relevant. Of these, 44 distinct genes were mutated in at least 2% of patients. The most common mutations (>7 patients) occurred in KRAS, NRAS, IGLL5, DIS3, BRAF, ACTG1, EGR1, FAM46C, TRAF3, DUSP2, FGFR3, and PRR14L. We also identified a deletion of IKZF3/Aiolos in a patient who progressed rapidly on lenalidomide-dexamethasone. Alterations in Ikaros family members like Aiolos have recently been reported as a potential mechanism of resistance to IMiDs. As the study continues to mature, we expect it will provide unprecedented molecular characterization and correlating clinical datasets that will help define the determinants of response to anti-myeloma agents and facilitate future clinical trial designs, thus serving as a stepping-stone toward personalized medicine for myeloma patients. Disclosures Lonial: Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca2+ pump isoform 4b (PMCA4b or ATP2B4) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in β4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway—by increasing the stability of PMCA4b—may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.

Abstract KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) constitute a group of cancer therapy agents designed to specifically disrupt and suppress hyperactive G protein signaling, such as that triggered by RAS mutations. Here we determine the effects of PCAIs on the viability, G-proteins levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutant, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 µM, respectively. To determine the effect of the PCAIs on the levels of Mitogen-Activated Pathway Kinase (MAPK)-related enzymes, western blot analysis was performed. After 48 hours exposure to 5 μM of the compounds, 57 to 150% increases of BRAF, ERK1/2, MEK1/2, and p90RSK phosphorylation along with a 60 to 78% decrease of pCRAF were observed, indicating significant disruptions in RAS signaling. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed 52 and 42% reduction in cell migration after 24 h exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. Western blot analysis was used to determine the effect PCAIs on caspase activation necessary for the initiation of apoptosis. After treatment with 5 μM of NSL-YHJ-2-27, full-length inactive caspase 3 and 7 levels dropped by 72 and 60%, while cleaved active caspase 3 and 7 levels increased by 274 and 130%, respectively. Treatment with 5 μM of NSL-YHJ-2-46 depleted full-length caspase 7 by 53% and increased cleaved active caspase 3 levels by 83%. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway, perhaps through the activation of proapoptotic isoforms of p90RSK. These results support the potential use of the PCAIs to serve as targeted therapies in cancers harboring RAS mutations. Citation Format: Matthew D. Gregory, Pablo E. Puente, Nadine L. Belony, Jassy Mary S. Lazarte, Nada Tawfeeq, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Treatment of a Black American lung cancer cell line harboring KRAS mutations with polyisoprenylated cysteinyl amide inhibitors reveals effects on the MAP kinase signaling pathway, cell migration and apoptosis [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C022.

The few therapeutic strategies for advance hepatocellular carcinoma (HCC) on poor knowledge of its biology. For several years, sorafenib, a tyrosine kinase inhibitors (TKI) inhibitor, has been the approved treatment option, to date, for advanced HCC patients. Its activity is the inhibition of the retrovirus-associated DNA sequences protein (RAS)/Rapidly Accelerated Fibrosarcoma protein (RAF)/mitogen-activated and extracellular-signal regulated kinase (MEK)/extracellular-signal regulated kinases (ERK) signaling pathway. However, the efficacy of sorafenib is limited by the development of drug resistance, and the major neuronal isoform of RAF, BRAF and MEK pathways play a critical and central role in HCC escape from TKIs activity. Advanced HCC patients with a BRAF mutation display a multifocal and/or more aggressive behavior with resistance to TKI. Moreover, also long non-coding RNA (lnc-RNA) have been studied in epigenetic studies for BRAF aggressiveness in HCC. So far, lnc-RNA of BRAF could be another mechanism of cancer proliferation and TKI escape in HCC and the inhibition could become a possible strategy treatment for HCC. Moreover, recent preclinical studies and clinical trials evidence that combined treatments, involving alternative pathways, have an important role of therapy for HCC and they could bypass resistance to the following TKIs: MEK, ERKs/ribosomal protein S6 kinase 2 (RSK2), and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR). These initial data must be confirmed in clinical studies, which are currently ongoing. Translational research discoveries could create new strategies of targeted therapy combinations, including BRAF pathway, and they could eventually bring light in new treatment of HCC.

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and &gt; 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values &lt; 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2674.

RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL), but expression of this mutation alone in murine B cells does not result in CLL. This gene mutation is often subclonal and associated with poor survival. How this mutation impacts CLL progression remains elusive. Since SF3B1 mutation frequently co-occurs with chromosome 13q deletion (del(13q)), and mice with deletion of the Minimal Deleted Region (MDR) of del(13q) develop indolent CLL, we therefore asked whether co-expression of Sf3b1 mutation can accelerate the onset of CLL in this murine model. If so, how does Sf3b1 mutation mechanistically contribute to CLL. To this end, we first crossed mice carrying conditional knock in allele Sf3b1-K700E and mice with conditional knockout of MDR. We then bred the offspring with CD19-Cre mice to generate cohorts of mice which have B cell-specific homozygous deletion of MDR with (DM) or without (MDR-MT) heterozygous Sf3b1-K700E. We monitored the onset of CLL by tracking of circulating B220+CD5+ CLL-like cells from peripheral blood with flow cytometry, starting at the age of 6-months and ending by 24-months. We detected CLL-like disease in 24% (6 of 25) of DM and 7.4% (2 of 27) of MDR-MT mice with disease presence in the spleen, bone marrow and lymph node, confirmed by flow cytometry and immunohistochemistry. The increased frequency of CLL in DM mice indicated that Sf3b1-K700E could accelerate CLL (Pearson Chi-Square 2-sided, p=0.098). To elucidate how Sf3b1 mutation contributes to increased CLL penetrance, we performed integrated RNA sequencing (RNA-seq) and TMT proteomics analysis with splenic B cells derived from DM mice with and without CLL. We found that genes involved in MYC, cell cycle checkpoints and mTORC1 pathways are upregulated and enriched at both the RNA and protein levels when we compared DM-CLL cells to their DM B cell counterparts, indicating these cellular processes are involved in the onset of CLL. To further define the role of Sf3b1-K700E mediated alternative splicing in the activation of these pathways, we first identified candidate splicing isoforms (nfatc1, braf, depdc5, tsc2) through computational analysis of RNA-seq data and then validated the isoforms in an independent cohort of samples (n=3,). Functional annotation of how exactly these isoforms impact CLL is ongoing. Importantly, we also observed gene upregulation of mTORC1 pathway in human CLL cells with SF3B1 mutation and del(13q) when compared with normal B cells. We next asked whether DM CLL cells are sensitive to inhibition of mTORC1 pathway and RNA splicing inhibition in vitro. We exposed DM B and CLL cells to either Temsirolimus (Tem, mTORC1 inhibitor), or H3B8800 (H3B, SF3B1 inhibitor) alone or in combination for 24 hours and then measured the cell viability with CellTiter-Glo assay. When compared to DMSO control, both Tem and H3B single treatments significantly inhibited the survival of DM CLL cells, but not DM B cells (all groups vs control, unpaired t test, p&lt;0.01). Furthermore, an additive effect was observed in DM CLL cells when 1nM of H3B was combined with Tem treatment (IC50: 1.2nM vs. 135.2uM, unpaired t test p&lt;0.001). We then tested the effects of both drugs in vivo using NSG mice engrafted with DM CLL cells. Mice treated with combination of Tem (15mg/kg, i.p, 5 days) and H3B (4mg/kg, gavage, 5 days) had a lower CLL burden in peripheral blood in comparison to either the single treatment or no drug treatment group (all groups vs. comb, p≤0.001). Furthermore, the combination treatment increased the survival of NSG mice engrafted with CLL cells compared to control (median survival: control vs. comb 15 vs. 34 days, log rank p&lt;0.001). Importantly, when we exposed human CLL cells with both del(13q) and sf3b1 mutation (DM-CLL, n=3), or with del(13q) alone (n=2), or normal B cells (n=4) to the combination treatment in vitro, DM-CLL cells showed the highest sensitivity to the treatment (DM-CLL vs. all groups, p&lt;0.05), suggesting that SF3B1 mutation may accelerate CLL with del(13q) through modulating RNA splicing and mTORC1 pathway. Our study demonstrates that expression of Sf3b1-K700E could accelerate the development of CLL based on MDR deleted murine model through alternative RNA splicing and mTORC1 activation. This finding supports the use of an mTORC1 inhibitor together with RNA splicing inhibitor in the subset of CLL patients with both SF3B1 mutation and del(13q). Disclosures Kipps: Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding. Neuberg:Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

9015 Background: The RAF inhibitors vemurafenib and dabrafenib (D) and the MEK inhibitor trametinib (T) improve survival as monotherapies in BRAF-mutant melanoma. Since clinical mechanisms of resistance (MoR) result in MAPK pathway reactivation, recent efforts have focused on combined targeting of RAF and MEK. The combination of D and T (D/T) increased progression-free survival and response rate compared with D alone (Flaherty et al, NEJM, 2012). The MoR to this combination remain unknown. Methods: To look for clinical MoR to combined RAF/MEK inhibition, we performed whole exome (WES) and whole transcriptome sequencing (RNASeq) on tumors from 4 patients (pts) with acquired resistance and 1 pt with intrinsic resistance to D/T. Pre-treatment and post-resistance tumors from all pts were analyzed for point mutations, insertions/deletions, copy number alterations, alternatively spliced transcripts, rearrangements, and expression changes. Results: In 2 of 4 pts with acquired resistance, WES identified mutations in MEK1 and MEK2 that were undetectable in the pre-treatment tumors. In the 3rd pt, RNASeq identified an alternatively spliced isoform of BRAF lacking exons 2-10, also undetectable in the pre-treatment tumor. In the 4th pt, no obvious MoR were seen, though multiple alterations were enriched in the post-resistance tumor. The pt with intrinsic resistance had several alterations in genes that conferred resistance to RAF/MEK inhibition when overexpressed in BRAF-mutant cell lines. Integration of WES and RNASeq data also identified several co-existing alterations that may synergize to increase resistance. Conclusions: Analysis of combined WES and RNASeq data from pt samples provides a more complete picture of clinical MoR to MAPK-targeted therapy. Post-resistance tumors from 3 of 4 pts with acquired resistance to D/T had alterations in MAPK genes not detectable in the pre-treatment tumors, suggesting that resistance involves reactivation of the MAPK pathway despite combined RAF/MEK inhibition. Alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

e15099 Background: NRAS mutations which activate MAPK signaling represent oncogenic driver alterations in approximately 20% melanoma cases in the US. NRAS mutant melanomas are uniquely dependent upon CRAF rather than BRAF for activation of downstream MEK/ERK signaling. In BRAF mutant melanoma, approved RAF-targeted therapies are commonly used in combination with a MEK inhibitor which provides clinical benefit by inhibition of two targets within the oncogenic MAPK signaling pathway. Emerging data with pan-RAF inhibitors in early clinical development suggests benefit with and without combined MEK inhibition, yet no approved targeted therapy exists for NRAS mutant melanoma patients. KIN-2787 is a novel, orally available pan-RAF inhibitor designed to be effective in RAF-dependent cancers, regardless of isoform. Methods: Kinome profiling was evaluated by radiometric enzyme assay at Reaction Biology across 688 kinases (including wild type, atypical, and mutant). Cellular activity was assessed by suppression of downstream MAPK pathway signaling and cell growth inhibition in human tumor cell lines. Combination cell growth inhibition studies were performed in 9x5 dose matrices with KIN-2787 and binimetinib, respectively. Extended cell growth inhibition effects were assessed by Incucyte imaging. In vivo KIN-2787 and combination efficacy was evaluated in NRAS mutant xenograft models. Results: Kinome profiling of KIN-2787 revealed exquisite selectivity with only 2 of 669 non-RAF family kinases inhibited > 75% at 1 M KIN-2787 and retained ̃10x and 70x selectivity window against those two kinases, DDR1 and p38b, respectively, relative to RAF kinases. We previously reported KIN-2787 activity across BRAF, NRAS, and KRAS mutant tumor cell lines with greatest sensitivity in Class II and III dimer-driven BRAF models. Here, we evaluated NRAS mutant, BRAF WT melanoma for combination potential with binimetinib. Melanoma tumor cell lines bearing NRAS hotspot mutations demonstrated synergistic benefit with KIN-2787 combined with binimetinib. Daily KIN-2787 plus binimetinib treatment in NRAS mutant melanoma xenograft models resulted in significant tumor growth inhibition benefit relative to either agent alone and was associated with added MAPK pathway biomarker suppression. Conclusions: KIN-2787 is a highly selective, potent, next-generation, pan-RAF inhibitor with activity across BRAF and RAS mutant human tumor cell models. Preclinical in vitro and in vivo studies using KIN-2787 in combination with binimetinib demonstrated significant combination benefit in NRAS mutant melanoma models. Taken together with its unique selectively, these data support use of KIN-2787 in combination therapy in this patient segment. A Phase 1/1b dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285).

e15099 Background: NRAS mutations which activate MAPK signaling represent oncogenic driver alterations in approximately 20% melanoma cases in the US. NRAS mutant melanomas are uniquely dependent upon CRAF rather than BRAF for activation of downstream MEK/ERK signaling. In BRAF mutant melanoma, approved RAF-targeted therapies are commonly used in combination with a MEK inhibitor which provides clinical benefit by inhibition of two targets within the oncogenic MAPK signaling pathway. Emerging data with pan-RAF inhibitors in early clinical development suggests benefit with and without combined MEK inhibition, yet no approved targeted therapy exists for NRAS mutant melanoma patients. KIN-2787 is a novel, orally available pan-RAF inhibitor designed to be effective in RAF-dependent cancers, regardless of isoform. Methods: Kinome profiling was evaluated by radiometric enzyme assay at Reaction Biology across 688 kinases (including wild type, atypical, and mutant). Cellular activity was assessed by suppression of downstream MAPK pathway signaling and cell growth inhibition in human tumor cell lines. Combination cell growth inhibition studies were performed in 9x5 dose matrices with KIN-2787 and binimetinib, respectively. Extended cell growth inhibition effects were assessed by Incucyte imaging. In vivo KIN-2787 and combination efficacy was evaluated in NRAS mutant xenograft models. Results: Kinome profiling of KIN-2787 revealed exquisite selectivity with only 2 of 669 non-RAF family kinases inhibited > 75% at 1 M KIN-2787 and retained ̃10x and 70x selectivity window against those two kinases, DDR1 and p38b, respectively, relative to RAF kinases. We previously reported KIN-2787 activity across BRAF, NRAS, and KRAS mutant tumor cell lines with greatest sensitivity in Class II and III dimer-driven BRAF models. Here, we evaluated NRAS mutant, BRAF WT melanoma for combination potential with binimetinib. Melanoma tumor cell lines bearing NRAS hotspot mutations demonstrated synergistic benefit with KIN-2787 combined with binimetinib. Daily KIN-2787 plus binimetinib treatment in NRAS mutant melanoma xenograft models resulted in significant tumor growth inhibition benefit relative to either agent alone and was associated with added MAPK pathway biomarker suppression. Conclusions: KIN-2787 is a highly selective, potent, next-generation, pan-RAF inhibitor with activity across BRAF and RAS mutant human tumor cell models. Preclinical in vitro and in vivo studies using KIN-2787 in combination with binimetinib demonstrated significant combination benefit in NRAS mutant melanoma models. Taken together with its unique selectively, these data support use of KIN-2787 in combination therapy in this patient segment. A Phase 1/1b dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285).

This work reports endoglucanase and beta-glucosidase production by the thermophilic fungusMyceliophthora heterothallicain solid-state (SSC) and submerged (SmC) cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. Forβ-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSCβ-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl3exerted the strongest inhibition for endoglucanases andβ-glucosidases.

The evolution of melanoma, the most aggressive type of skin cancer, is triggered by driver mutations that are acquired in the coding regions of particularly BRAF (rat fibrosarcoma serine/threonine kinase, isoform B) or NRAS (neuroblastoma-type ras sarcoma virus) in melanocytes. Although driver mutations strongly determine tumor progression, additional factors are likely required and prerequisite for melanoma formation. Melanocytes are formed during vertebrate development in a well-controlled differentiation process of multipotent neural crest stem cells (NCSCs). However, mechanisms determining the properties of melanocytes and melanoma cells are still not well understood. The nerve growth factor receptor CD271 is likewise expressed in melanocytes, melanoma cells and NCSCs and programs the maintenance of a stem-like and migratory phenotype via a comprehensive network of associated genes. Moreover, CD271 regulates phenotype switching, a process that enables the rapid and reversible conversion of proliferative into invasive or non-stem-like states into stem-like states by yet largely unknown mechanisms. Here, we summarize current findings about CD271-associated mechanisms in melanoma cells and illustrate the role of CD271 for melanoma cell migration and metastasis, phenotype-switching, resistance to therapeutic interventions, and the maintenance of an NCSC-like state.

Liquid biopsy has emerged as an alternative source of nucleic acids for the management of Epidermal Growth Factor Receptor (EGFR)-mutant non-Small Cell Lung Cancer (NSCLC). The use of circulating cell-free DNA (cfDNA) has been recently introduced in clinical practice, resulting in the improvement of the identification of druggable EGFR mutations for the diagnosis and monitoring of response to targeted therapy. EGFR-dependent (T790M and C797S mutations) and independent (Mesenchymal Epithelial Transition [MET] gene amplification, Kirsten Rat Sarcoma [KRAS], Phosphatidyl-Inositol 4,5-bisphosphate 3-Kinase Catalytic subunit Alpha isoform [PI3KCA], and RAF murine sarcoma viral oncogene homolog B1 [BRAF] gene mutations) mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been evaluated in plasma samples from NSCLC patients using highly sensitive methods (i.e., digital droplet PCR, Next Generation Sequencing), allowing for the switch to other therapies. Therefore, liquid biopsy is a non-invasive method able to detect the molecular dynamic changes that occur under the pressure of treatment, and to capture tumor heterogeneity more efficiently than is allowed by tissue biopsy. This review addresses how liquid biopsy may be used to guide the choice of treatment strategy in EGFR-mutant NSCLC.

468 Background: Tumor cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase (PKM2), which is a key enzyme that regulates aerobic glycolysis in tumor cells. Use of RNA interfering (RNAi) targeting PKM2 significantly inhibits tumor growth when combined with cisplatin in xenograft models. We evaluated the predictive significance of PKM2 in patients with “high” risk stage II or stage III colon cancer treated with oxaliplatin-fluoropyrimidines chemotherapy adjuvant treatment. Methods: FFPE primary tumours from 261 patients with stage II or III colon cancer with colon cancer were analyzed for PKM2 mRNA expression by RT-qPCR. All patients had received adjuvant treatment with an oxaliplatin and fluoropyrimidine combination. Also, match normal mucosas from 50 of these patients have been analyzed. Finally, 50 polyps (20 with low grade dysplasia, 20 high grade dysplasia and 10 serrated adenomas) from non-colon cancer patients were included in the for PKM2 mRNA expression by RT-qPCR. The analysis was performed with a set of primers and probes which amplify only the M2 isoform. Results: PKM2 mRNA expression was significantly higher in tumor areas in comparison with normal mucosa (p=0.001) and low-grade adenomas (p=0.001) as well as in tumors with BRAF mutations (p=0.001). Adenomas with high-grade dysplasia (p=0.002) and serrated adenomas (p=0.001) presented significantly higher PKM2 mRNA expression. Overexpression of PKM2 was associated with significantly lower Disease Free (p=0.018) and Overall Survival (p=0.031). Multivariate analysis revealed PKM2 high mRNA expression as independent prognostic factors for decreased DFS (HR: 2.1, p=0.003) and OS (HR: 1.8, p=0.019). Conclusions: These results provide evidence that the PKM2 mRNA expression could be used as prognostic/predictive marker for disease recurrence in patients with operable colon cancer. Analysis of the PKM2 mRNA expression in patients treated with non-oxaliplatin containing adjuvant treatment is currently performed.

Abstract Background. Progression to acute myeloid leukemia (sAML) occurs in 20% of myelofibrosis (MF) and 10% of polycythemia vera (PV) or essential thrombocythemia (ET). sAML has dismal outcome with median survival of <6 months. We recently reported that a restricted set of mutations predict for leukemic transformation in MPN (Vannucchi AM, Leukemia 2013; Tefferi A, Blood Adv. 2016). However, the molecular mechanisms underlying transformation to sAML are poorly characterized, in particular the relationships between the clones establishing the chronic phase and the one dominating the leukemic phase. Aim. To study clonal heterogeneity and clonal progression in the leukemic transformation of MPN we performed whole exome (WES) and trascriptome (RNAseq) sequencing of paired samples (chronic (CP)/blast phase(BP). Patients and Methods. WES and RNAseq was done on CD34+ or blast cells of 12 paired samples from 5 PMF, 3 PET-MF, 1 PPV-MF, 3 ET pts who transformed to sAML. In addition, a myeloid neoplasm-relevant 34-gene panel was used for target NGS sequencing of paired samples plus an additional 23 samples collected from MPN pts at leukemic phase (15 PMF, 3 PET-MF, 3 ET, 2 PV). Results. In the entire series, 20 pts (57%) were JAK2 V617F mutated at CP, 8 CALR (23%; 6 type-1 and 2 type-2) and 7 (8.6%) MPL mut, while 4 (11.4%) were triple-negative. JAK2 variant allele frequency (VAF) declined by ≥50% (n=3; 15%) or became undetectable (n=4; 20%) whereas 3 heterozygous pts (15%) became homozygous at the time of BP. One of the 8 CALR mut cases halved its VAF, whereas no meaningful VAF changes were observed in MPL mut pts. The most frequent mutated genes detected by NGS at BP, other than driver mutations, were ASXL1 (51.4%), RUNX1 (37.1%), TET2 (17%), SRSF2 (16%), IDH1 (14%), TP53 (14%), NRAS (14%), FLT3 (14%), U2AF1 (11%) and KRAS (11%); three (8%) cases were mutated for EZH2DNMT3A, CBL and PTPN11, 2 cases for IDH2, SH2B3, SF3B1 IKZF1, SETBP1 and ZRSR2, and 1 case for ABL1, ATV6, BRAF and ARID1A. There were also 5 CEBPA-mutated pts, 3 of which, unlike none of 27 CEBPA-mutated de novo AML, showed an in-frame 6-bp duplication polymorphism (p. P196_197insHP) reported in 20-40% of all de novo AML and <1% of healthy population (rs762459325). Compared to the CP of the 12 paired samples, 10 pts (83%) acquired novel mutations, 3 of which acquired ≥3 variants. RUNX1 and ASXL1 were those with the highest number of acquisition (n=7 and 5, respectively). Conversely, loss of variants was observed only in ASXL1 (n=3). By WES analysis, on average 60.000 variants per paired sample unique to the BP compared with CP were identified. However, no recurrent abnormalities were found in the 12 paired samples outside the above listed mutations found by target sequencing. For RNAseq analysis, transcripts were annotated according to UCSC hg19 for a total of 25,369 genes. We found large transcriptional differences between CP and BP with 129 transcripts differentially expressed (23 up- and 106 down-regulated) and 4,155 isoforms (2,120 up- and 2,035 down-regulated). Among the most abnormally expressed transcripts we selected 8 genes (5 down-regulated: LCN2, PDGFB, PRTG, CRISP3, PF4; 3 up-regulated CDKN2, SH2D1A and LIN28) for validation by QRT-PCR based on the extent of differential expression. PF4 and CDKN2 were confirmed to be down-regulated and over-expressed in BP, respectively (P<0.0001). Analysis of RNA-seq data for fusion genes revealed 10 fusion genes acquired during BP; however, only 3 of them were confirmed by Sanger sequencing (2 cases of BCR-ABL, 1 case of KMT2A- MLLT3 and 1 case of CBFB-MYH11). Pathway analysis included Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. Six pathways were significantly deregulated in BP compared with CP: Mitotic Spindle (P<0.0001); Androgen response (P=0.004); TGF-beta signaling (P=0.04); Apoptosis (P=0.03); PI3K-AKT-MTOR signaling (P=0.04); Reactive Oxygen Species- Pathway (P=0.03). Conclusions. Comprehensive comparative analysis of genomic and RNA abnormalities acquired in the transition from CP to BP in MPN has not been previously reported. Our data indicate that BP is associated with significant changes in mutation complexity and RNA expression, overall affecting different intracellular pathways whose further characterization might help to identifying potential targets for therapy. Disclosures No relevant conflicts of interest to declare.

Abstract TAR is a rare bone marrow failure syndrome comprised of thrombocytopenia and a spectrum of bony abnormalities, the most common being bilateral radial aplasia in the presence of thumbs. Recent research has implicated alterations in the RBM8A gene in the pathogenesis of this disorder. Diffuse LCH is a neoplasm of mature Langerhans cells, a subset of dendritic cells, that has now been demonstrated to be clonal in nature. The co-occurrence of these two rare disorders has only been reported twice (Ingram, BMT 2006; Guastadisegni EJMG 2012). We report the third case of a patient with TAR and LCH and present the molecular and genetic characterization of this patient. A 52 year woman with TAR presented with abdominal mass, splenomegaly and left axillary adenopathy. CT scan showed a right pelvic mass measuring 11.5 x 8.6 cm, retroperitoneal lymphadenopathy, enlarged spleen and patchy areas of radiolucency throughout the spine, pelvis and left femoral head. Excisional biopsy of the left axillary lymph node revealed complete effacement of the normal lymph node architecture by an atypical proliferation of Langerhans cell histiocytes. Bone marrow involvement was documented as well. On immunohistochemistry, these cells were positive for CD1a (membrane staining) and S-100 (nuclear and cytoplasmic staining). As part of the diagnostic evaluation, CGH microarray (Signature Genomics) was performed. A diagnosis of diffuse LCH was made. She was treated with vinblastine and prednisone for a year and remains in complete remission from her disease 14 months later. Genomic DNA was extracted from peripheral blood (normal mononuclear cells) and paraffin-embedded tumor tissue, with written informed consent following the approval of the Institutional Review Board of Thomas Jefferson University. The CGH result on the patient's uninvolved tissue as well as the tumor sample showed a hemizygous deletion of 1q21.1 encompassing the RBM8A gene, consistent with recent TAR literature (Albers, Nat Genet 2012). Sequence analysis of the other RBM8A allele revealed minor allele A at SNP rs139428292 at position chr1:145,507,646 (hg19) in the 5'UTR of RBM8A and nt G at chr1:145,507,777 in the first intron (int1 +44). The coding sequence was normal. Minor allele A at the 5'UTR SNP has been associated with TAR syndrome, along with minor allele C at intronic SNP (rs 201779890, int1 +32) which was major allele G in our patient. However, nt G at intron 1 +44 is a novel variant that has not been reported before in dbSNP137, HapMap or 1000 Genomes (UCSC Genome Browser, August 1, 2013). The CGH result on the patient's tumor sample showed a 1.82 Kb deletion within the MAF gene. We identified in normal and tumor DNA one intact MAF gene and one deleted of all of the coding sequences of MAF isoform 1 (alternatively, complete deletion of exon 1 of MAF isoform 2). In addition, a copy loss in the 14q32.2 region was found in the tumor sample but not in the uninvolved tissue. This region contains the BCL11b gene. Finally, we identified a tumor-specific missense mutation (GTG to GAG) resulting in a change in amino acid from valine to glutamine at codon 600. This is the BRAF V600E mutation previously described in LCH and other malignancies. In summary, we report the third ever reported case of the co-occurrence of two rare disorders, TAR and LCH, in a single patient and identify novel molecular changes. We have found the patient to have the recently reported RBM8A genetic alterations of TAR (microdeletion of 1q21 for one allele, minor 5’UTR SNP on the other allele), plus a novel RBM8A intronic SNP. We found a somatic MAF deletion in our patient, possibly a predisposing factor towards LCH. In addition, BRAF V600E mutation is a tumor-specific mutation in our patient. Whether BRAF inhibitors will be of therapeutic benefit in primary or relapsed LCH is currently unknown. Future research is needed to elucidate the function, if any, of the novel RBM8A SNP, the mechanism(s) by which the identified SNPs lead to reduced Y14 levels, and the potential contribution of Y14 deficiency in tumorigenesis. The authors wish to acknowledge the invaluable role of Dr. Elena Gitelson in the care of this patient. Disclosures: No relevant conflicts of interest to declare.

Wheat is an important cereal with a key role in human nutrition. In this study, dietary fiber (DF) and arabinoxylans of different durum (Triticum turgidum ssp. Durum L.) and bread (Triticum aestivum L.) wheat flours were analyzed in order to point out their potential nutritional and health claims allege according to the current European regulation (Regulation (EU) No 432/2012). Moreover, other bioactive compounds (phenolics and tocopherols) were quantified as a first approach to their phytochemical composition in the analyzed wheat varieties. DF was analyzed following AOAC enzymatic-gravimetric methods; arabinoxylans and total phenols were quantified by colorimetric methods; tocopherols were determined by HPLC; antioxidant activity was evaluated using three different in vitro assays. Insoluble DF was the prevailing fraction in all analyzed samples. Water extractable arabinoxylans were higher in durum wheat flours. Whole flours contained higher total phenolics compounds. Alpha-tocopherol was the major isoform. Whole flours showed higher antioxidant properties. According to the obtained results, it is possible to allege all approved health claims referred to wheat, since all analyzed samples, especially whole flour and bran fraction, showed potential health benefits, as functional ingredients or functional foods, related with their phytochemical composition.

Összefoglaló. A molekuláris onkológia térnyerésével számos új lehetőség érhető el a daganatos betegek hatékonyabb kezelésére. Ilyen a klinikai vizsgálatokban alkalmazott, a valóban személyre szabott kezelést elősegítő génpanelelemzés, illetve a célzott kezelés szövettípustól független alkalmazása. A személyre szabott terápiák jelentős hányada valamelyik kinázt gátolja. Az összefoglalónkban bemutatjuk a RAS jelátviteli út sejten belüli komplex szabályozását, valamint ismertetjük az útvonal további farmakológiai szempontból kiaknázható célpontjait nemzetközi és saját eredményeink alapján. A kinázokat érintő gyakori mutációk ellenére számos daganattípusban nem áll rendelkezésre személyre szabott terápia. A hagyományos terápiával nem kezelhető agydaganatok példáján keresztül bemutatjuk a tirozin-kinázok várható jövőbeli terápiás jelentőségét. Summary. With the advent of molecular oncology, the identification of mutations in solid tumours is now clinically routine. The growing repertoire of targeted therapeutic agents has supported the rise of a new type of clinical trial in which the selection of the therapeutic agent is no longer restricted to a single option. Instead, a panel of genes is screened to identify the most suitable drug for each patient. Such trials have delivered objective response rates in 5–30% of patients. Most of the signal transduction pathways targeted by these agents involves RAS signaling. Somatic mutations in RAS genes are common in human tumours. Such mutations generally decrease the ability of RAS to hydrolyze GTP, maintaining the protein in a constitutively active GTP-bound form that drives uncontrolled cell proliferation. Recent emerging data suggest that RAS regulation is more complex than the scientific community has appreciated for decades. We discuss a novel type of RAS regulation that involves direct phosphorylation and dephosphorylation of RAS tyrosine residues. The discovery that pharmacological inhibition of the tyrosine phosphoprotein phosphatase SHP2 maintains mutant Ras in an inactive state suggests that SHP2 could be a novel drug target for the treatment of Ras-driven human cancers. In addition to RAS gene mutations, other common oncogenic events have also been identified, including mutation of EGFR (epidermal growth factor receptor) or BRAF (isoform B of rapidly accelerated fibrosarcoma). EGFR has tyrosine kinase activity while BRAF acts as a serine/threonine kinase. In some tumours, mutant forms of these kinases over-activate cell proliferation, leading to uncontrolled tumour cell growth; therefore, it seems rational to develop inhibitor molecules that target these hyper-active oncogenic kinases to reduce tumour cell burden in cancer patients. Fusion protein kinases activated via the RAS pathway represent target proteins with high clinical success rates. Recently approved TRK fusion protein kinase inhibitors have reached response rates in almost 80% of patients regardless of tumour type. Although these drugs can only be administered to patients whose tumours harbour a TRK fusion protein, such success stories pave the way for future development of agents that target similar genetic mutations. Glioblastoma multiforme, a relatively frequent, almost uniformly fatal brain tumour, has ubiquitous alterations in tyrosine-kinase signalling. Nevertheless, to this day, no tyrosine-kinase inhibitors have been approved for its treatment. We have ongoing research projects to uncover associations between initial gene expression levels in untreated glioblastoma samples and treatment-related survival, and we have identified overexpression of druggable tyrosine-kinase receptors in chemotherapy-resistant patients. Our approach will help to identify patients who might benefit from concurrent use of tyrosine kinase inhibitors and conventional cytotoxic therapies.

Abstract The fibroblast growth factor receptor (FGFR) family of protein tyrosine kinases plays a role in many physiological processes including angiogenesis and wound healing. FGFR mutations, fusions, rearrangements, and amplifications have been linked to the pathogenesis of multiple tumor types. For example, approximately 10-15% of patients with intrahepatic cholangiocarcinoma have FGFR2 fusions, and amplification of FGFR2 is prevalent in gastric cancer and implicated in tumor growth. Approved FGFR inhibitors produce responses in patients that harbor FGFR genetic alterations but show reduced activity against key mutations (e.g., V565, N550, etc.). These pan-FGFR inhibitors also frequently show dose-limiting toxicities including hyperphosphatemia which can be linked to the inhibition of FGFR1. To address these issues, a novel series of FGFR inhibitors that are potent and selective reversible inhibitors of FGFR2 with selectivity over FGFR1 have been identified. This series also maintains activity against clinically relevant mutations in the FGFR2 protein. Inhibition of cellular FGFR phosphorylation was measured using engineered cell lines to demonstrate that the inhibitors target both wild-type FGFR2 and activating FGFR2 mutations while sparing FGFR1. In addition, CDX tumor models showed in vivo target engagement and FGFR isoform selectivity. Herein, the in vitro and in vivo characterization of a representative selective reversible FGFR2 inhibitor is described. Citation Format: John Fischer, Karyn Bouhana, Mark J. Chicarelli, Josh Dahlke, Brad Fell, Jennifer Fulton, Anna Guarnieri, Ravi Jalluri, Amber Johnson, Brent Mclean, Max Mejia, Rob Rieger, John Robinson, Mareli Rodriguez, Francis Sullivan, Yang Wang, Shannon Winski, Yeyun Zhou. Pre-clinical characterization of a novel series of FGFR2 selective inhibitors with potency against clinically relevant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 167.

Transformer 2 (tra 2) and fruitless (fru) genes have been proven to play a key role in sex determination pathways in many Arthropods, including insects and crustaceans. In this study, a paralog of P. monodon tra 2 (Pmtra 2), P. monodon ovarian associated transformer 2 (PmOvtra 2) and 2 isoforms of P. monodon fruitless-like gene (Pmfru-1 and Pmfru-2) were identified and characterized. The full cDNA sequence of PmOvtra 2 consisted of 1,774 bp with the longest open reading frame (ORF) of 744 bp encoding for 247 amino acids. The PmOvtra 2 exhibited a predicted RNA-recognition motif (RRM) domain and two arginine-serine (RS) regions, suggesting its function in RNA splicing. The full cDNA sequence of Pmfru-1 consisted of 1,306 bp with 1,182 bp ORF encoding for 393 amino acids, whereas the full cDNA sequence of Pmfru-2 consisted of 1,858 bp with 1,437 bp ORF encoding 478 amino acids. The deduced amino acid sequences of Pmfru-1 and Pmfru-2 exhibited highly conserved domains of Fru proteins, including Broad-complex, Tramtrack and Bric-a-brac (BTB), and zinc finger (ZF) domains. In addition, Pmfru-1 and Pmfru-2 were suggestively originated from the same single genomic locus by genomic sequence analysis. Specifically, Pmfru pre-mRNA was alternatively spliced for Pmfru-1 and Pmfru-2 to include mutually exclusive exon 7 and exon 6, respectively. Temporal and spatial expression of PmOvtra 2, Pmfru-1, and Pmfru-2 were also investigated by qPCR. The results showed that all were expressed in early developmental stages with undifferentiated gonads starting from nauplius until postlarvae. The expression of PmOvtra 2 started at nauplius stage and gradually increased from mysis to postlarvae (PL) 1. However, the expression of Pmfru-1 was low at the nauplii stage and slightly increased from protozoea to PL5, whereas the expression of Pmfru-2 maintained a low level from nauplius to mysis and then gradually increased at the PL stages. Expressions of PmOvtra 2, Pmfru-1, and Pmfru-2 were detected in various tissues including nervous tissue, gill, heart, hepatopancreas, gut, and gonads. Interestingly, the sexually dimorphic expression of PmOvtra 2, Pmfru-1, and Pmfru-2 was demonstrated in fully developed gonads in which the ovary showed significantly higher expressions than the testis. The great difference in the expression pattern of PmOvtra 2, Pmfru-1, and Pmfru-2 in the ovary and testis suggested their roles in the female sex determination in P. monodon.

Background:Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective treatment in selected patients with autoimmune diseases. AutoHSCT can induce long-term remission with 80% progression free survival in multiple sclerosis patients (Muraro 2017, Burt 2019). Use of autoHSCT in scleroderma patients has achieved superior outcomes in two randomized studies compared to standard of care (Tyndall 2014, Sullivan 2018). These impressive results are achieved by a combination of the eradication of autoreactive immune effector cells and re-establishment of self-tolerance, i.e., immune system reset. However, only a small fraction of eligible patients undergo autoHSCT, largely due to toxicity associated with current conditioning protocols.Objectives:As part of our goal to enable more patients to benefit from immune system reset, we have generated novel anti-human CD45 ADCs that cross react with nonhuman primates (NHP) and an anti-mouse CD45 ADC to model the approach in mouse models of AID.Methods:The human-targeted CD45-ADC is an affinity-matured mAb that targets an epitope present on all human CD45 isoforms, is cross-reactive with NHP CD45, and is conjugated to a payload that efficiently kills both quiescent and cycling cells. This ADC is engineered to eliminate Fc-mediated effector function, enable site-specific conjugation of linker/payload, and enable rapid clearance. This ADC was evaluated in vitro and in vivo in hNSG and NHPs. The murine tool ADC specifically targets the CD45.2 isoform of mouse CD45, and is also engineered to eliminate effector function, allow for site-specific conjugation of linker payload, and be rapidly cleared. The payload for this murine tool ADC is potent and preferentially kills dividing cells. This ADC was tested for the ability to enable immune reset and ameliorate autoimmune disease in multiple disease models.Results:The anti-human CD45-ADC showed efficient killing of human HSCs and human and cyno PBMC, including CD3+cells from healthy donors and patients with MS. In hNSG, single doses of the CD45-ADC were well-tolerated and led to substantial depletion of human cells. In NHPs, single doses of CD45-ADC were well tolerated and depleted both peripheral lymphocytes and HSCs. Administration of a single dose of anti-human CD45-ADC to hNSGs with sclerodermatous xenoGVHD resulted in depletion of human T cells and resolution of symptoms. A single-dose of the anti-mouse CD45-ADC enabled full myeloablation and complete durable donor chimerism with congenic HSCT at 16 weeks. In a murine immunization model of MS, MOG-induced EAE, a single dose of the CD45-ADC followed by congenic HSCT prior to disease onset enabled full donor chimerism, significantly delayed disease onset and reduced disease severity. We are generating additional data in an adoptive transfer model of EAE to confirm and extend these results. In a murine model of arthritis, therapeutic treatment with a single dose of the CD45-ADC followed by congenic HSCT enabled complete donor chimerism and halted disease progression, comparable to with the effects of an anti-TNFα antibody. The ADC is being further evaluated in a model of type 1 diabetes and those data will be presented. These data demonstrate that CD45-ADC conditioning followed by congenic HSCT is sufficient for full myeloablation and immune reset.Conclusion:These results demonstrate that targeted immune depletion with a single dose of CD45-ADC can enable auto-HSCT and immune reset in multiple AID indications without toxic side effects. Targeted conditioning with CD45-ADC may represent a better tolerated approach for removing disease-causing cells as part of immune reset through auto-HSCT and enable more patients to benefit.Disclosure of Interests:Geoffrey Gillard Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Jennifer Proctor Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Sharon Hyzy Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Oliver Mikse Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Tahirih Lamothe Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Sean McDonough Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Nicholas Clark Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Rahul Palchaudhuri Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Anjali Bhat Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Melissa Brooks Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Ganapathy Sarma Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Prashant Bhattarai Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Pranoti Sawant Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Brad Pearse Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Charlotte McDonagh Shareholder of: Magenta Therapeutics, Employee of: Magenta Therapeutics, Tony Boitano Shareholder of: Magenta, Employee of: Magenta, Michael Cooke Shareholder of: Magenta, Employee of: Magenta

Abstract Monoclonal antibodies (MAbs) are a well-established treatment approach in oncology and other diseases. Nevertheless, many multipass membrane proteins are largely inaccessible as antibody targets due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. Integral Molecular’s MPS Antibody Discovery platform specifically addresses each of these challenges. A key enabling feature of MPS is the use of chickens as an evolutionarily divergent host species for immunization, allowing a more robust immune response for targets that are highly conserved in mammals. We will present on antibodies isolated against challenging membrane protein targets in oncology including Claudin 18.2 (CLDN18.2).CLDN18.2 is a transmembrane adhesion protein undetectable in most adult healthy tissues but highly expressed in gastric, pancreatic, esophageal, and lung cancers. Antibody discovery efforts against this validated target are challenging due to the abundant expression of the splice isoform CLDN18.1 differing by only 8 amino acids in the extracellular domain. As part of the MPS platform, we used virus-like particles (Lipoparticles) to immunize chickens with a high concentration of native CLDN18.2 protein and obtain high-titer immune responses. This enabled us to generate and isolate a large and diverse collection of MAbs (48 unique clones) and select candidates for optimization. We present a panel of three highly specific, humanized CLDN18.2 MAbs with picomolar affinities that are superior to the clinical-stage benchmark. We will show in-depth profiling data for the MAb panel that were used for lead selection and de-risking clinical development. These data include biosensor binding kinetics, amino-acid resolution epitope mapping, and specificity testing against the Membrane Proteome Array (MPA) consisting of 6,000 membrane proteins. This panel of preclinical antibodies are being developed for therapeutic use in various formats, including bispecifics, antibody-drug conjugates, and CAR-T applications. Citation Format: Brad Screnci, Trevor Barnes, Kristen Shema, Rebecca Rimkunas, Shruthi Kannan, Tim Phillips, Carmen Navia, Charles Azuelos, Tom Charpentier, Jennifer Houtmann, Lisa Miller, Lewis J. Stafford, Benjamin J. Doranz, Joseph B. Rucker, Ross Chambers. Isolation of highly selective antibodies against claudin 18.2 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 317.

Introducción: La enfermedad renal crónica asociada a la obesidad (ERC-AO) es una enfermedad con aumento en la prevalencia en las últimas décadas. Se caracteriza por un exceso de desequilibrios hormonales adipocíticos (adipoquinas), desregulación del sistema de equilibrio energético y desequilibrios en la homeostasis metabólica. Propósito de la revisión: El objetivo de la revisión es delinear el papel de los diferentes mecanismos fisiopatológicos para el desarrollo de enfermedad renal funcional o anatómica en pacientes con obesidad. Buscamos reportes actualizados en donde se incluye los resultados de mejor supervivencia para los pacientes con ERC-AO. Recientes hallazgos: Actualmente sabemos la ERC-AO tiene un comportamiento pro inflamatorio crónico. La obesidad y sobrepeso se asocian alteraciones hemodinámicas, estructurales e histopatológicas en el riñón, así como alteraciones metabólicas y bioquímicas que predisponen a la enfermedad renal, aun cuando la función renal y las pruebas convencionales sean normales. Conclusiones: Clasificamos a la ERC-AO en Tipo 1: Obesidad y alteraciones funcionales potencialmente reversibles. Tipo 2: Obesidad y alteraciones estructurales histopatológicas potencialmente no reversibles (Incluye la Glomerulopatía asociada a obesidad y glomeruloesclerosis focal y segmentaria). Tipo 3: Obesidad en relacionada con enfermedades crónicas (Diabetes, Hipertensión, Hipertensión pulmonar. Insuficiencia Cardíaca). Tipo 4: Obesidad en el paciente con terapia sustitutiva de la función renal. Recibido: Agosto 03, 2022 Aceptado: Septiembre 30, 2022 Publicado: Septiembre 30, 2022 Editor: Dr. Franklin Mora Bravo. Introducción La obesidad es una enfermedad en crecimiento con un aumento en su prevalencia en las últimas décadas, asociándose a un elevada carga asistencial y económica para los sistemas sanitaros derivado de su relación con enfermedades cardiovasculares, endocrinas, psicológicas, renales entre otras [1, 2]. El incremento en las tasas de obesidad en distintos grupos etarios, desde niños hasta adultos jóvenes conlleva a asumir que en el futuro veremos más enfermedad renal relacionada con la obesidad (ERC-AO) en la población general, con implicaciones relevantes para los sistemas de atención [3]. Por ello el conocimiento y comprensión de esta interacción podría tener implicaciones en la prevención y tratamiento de las enfermedades renales. Dentro de la población general la obesidad se asocia a incremento en el riesgo de diversas condiciones patológicas, como la hipertensión arterial crónica (HTA), enfermedad renal crónica (ERC), artrosis, infecciones, síndrome de apnea hipopnea obstructiva del sueño (SAHOS) y diabetes mellitus (DM) entre otras [3]. No obstante, en el escenario de la ERC, la obesidad juega un rol dual y paralelo en el desarrollo de la enfermedad, tradicionalmente se ha denominado “paradoja de la obesidad”, donde por un lado actúa como un factor de riesgo modificable para el desarrollo de la enfermedad renal crónica (ERC) y por otro se ha asocia de manera consistente con mejores resultados de supervivencia en pacientes con enfermedad renal terminal [1]. Por lo anterior, en las próximas páginas describimos aspectos fisiopatológicos que involucran la obesidad en el desarrollo de la ERC. Definición y epidemiología La obesidad es una condición que se caracteriza por la acumulación anormal o excesiva de tejido adiposo con consecuencias patológicas adversas e incremento del riesgo cardiovascular [4]. Utilizando para su definición y diagnostico un indicador simple como es la relación entre el peso y la talla denominado índice de masa corporal (IMC), se calcula dividiendo el peso de una persona en kilos por el cuadrado de su talla en metros (kg/m2). Un IMC entre 18.5 y 25 kg/m2 es considerado por la Organización Mundial de la Salud (OMS) como peso normal, un IMC entre 25 y 30 kg/m2 como sobrepeso y un IMC > 30 kg/m2, como obesidad [5-7]. Además, la obesidad puede ser clasificada en tres niveles de severidad: clase I (IMC 30.0 – 34.9), clase II (IMC 35.0 – 39.9) y clase III (IMC > 40) [8]. Durante las últimas tres décadas, la prevalencia de adultos con sobrepeso y obesidad (IMC ≥ 25 kg/m2) en todo el mundo ha aumentado sustancialmente, convirtiendo a la obesidad en una epidemia y se prevé que su prevalencia crezca un 40% en la próxima década [6]. Actualmente, el problema de obesidad se ha visto en mayor aumento debido al incremento en la afectación en niños, lo que ocasiona una mayor prevalencia de patologías a edad temprana. En 2016, según las estimaciones de la OMS unos 41 millones de niños menores de cinco años tenían sobrepeso o eran obesos [7]. Esto afectando a todos los países, independiente de su nivel de ingresos [7]. La prevalencia del sobrepeso y la obesidad en niños y adolescentes (de 5 a 19 años) ha aumentado de forma espectacular, del 4% en 1975 a más del 18% en 2016. Este aumento ha sido similar en ambos sexos: un 18% de niñas y un 19% de niños con sobrepeso en 2016. Mientras que en 1975 había menos de un 1% de niños y adolescentes de 5 a 19 años con obesidad, en 2016 eran 124 millones (un 6% de las niñas y un 8% de los niños) [7]. La creciente prevalencia de la obesidad tiene implicaciones para las enfermedades cardiovasculares (ECV) y también para la ERC. Un IMC alto es uno de los factores de riesgo más fuertes para la ERC de nueva aparición [6]. Epidemiología de la enfermedad renal crónica asociada a obesidad (ERC-AO) La enfermedad renal crónica (ERC) es una condición de interés en salud pública, asociada a una elevada morbilidad y mortalidad a nivel mundial. Las guías KDIGO (Kidney Disease: Improving Global Outcomes), definen la ERC como la presencia de alteraciones en la estructura o función renal durante al menos tres meses y con implicaciones para la salud [9, 10]. Los principales elementos clasificatorios para definir la presencia de ERC son la tasa de filtración glomerular (TFG) estimada (G1 a G5) utilizando como umbral definitorio una TFG 60 ml/min/1,73m2 y la tasa de excreción de albúmina en orina (A1 a A3) según el cociente albúmina/creatinina en una muestra aislada de orina sea < 30, 30-300 o > 300 mg/g, respectivamente [9, 10]. Si bien inicialmente existía cierta controversia sobre el uso de la TFG para el diagnóstico de la ERC en fases iniciales, trabajos recientes han puesto en evidencia que tanto una TFG< 60 ml/min/1.73 m2 como un cociente albúmina/creatinina (CAC) ≥ 1.1 mg/mmol (10 mg/g) son predictores independientes del riesgo de mortalidad e insuficiencia renal terminal (IRT) en población general [11, 12]. En consecuencia, debido a estas categorías podemos determinar el pronóstico de cada paciente. Los datos globales sugieren que la prevalencia de la ERC se encuentra entre el 10 y el 16 %, pero la información sobre la prevalencia de la población por categoría de TFG y ACR es escasa [13]. La ERC es una afección asociada a una elevada carga de morbilidad, mortalidad y enfermedad cardiovascular (ECV). A medida que disminuye la función renal, surgen trastornos metabólicos y hemodinámicos que aumentan las tasas de hospitalización, ECV y muerte [4]. El conjunto de factores de riesgo conocidos para la progresión de la ERC es relativamente pequeño, y las terapias y estrategias efectivas para retrasar la progresión de la ERC son limitadas [14]. Por lo cual resulta necesario conocer y entender los diferentes factores de riesgo y su impacto en el daño renal, en aras de lograr minimizar la progresión del mismo, sobre todo en aquellos en los cuales se puede realizar intervenciones activas, evaluables, controlables y con seguimiento continuo como es la obesidad. A la fecha existe suficiente evidencia para asociar la obesidad con el desarrollo y progresión de la enfermedad renal crónica. Los datos granulares sobre la prevalencia de la obesidad en personas con ERC son limitados pero consistentes en todo el espectro de la enfermedad renal. En la Encuesta Nacional de Examen de Salud y Nutrición de 2011–2014, el 44.1 % de los pacientes con ERC en los Estados Unidos también tenían obesidad (21.9 % con obesidad de clase 1 y 11.1 % con clase 2 y obesidad clase 3, habiéndose incrementado el porcentaje global un 5% en los últimos 12 años [15]. La glomeruloesclerosis focal y segmentaria (GEFS) es el tipo de glomerulonefritis que se asocia con mayor frecuencia a la obesidad [16]. La enfermedad glomerular habitualmente asociada a la obesidad se denomina glomerulopatía relacionada con la obesidad (GRO). Esta condición suele presentarse con síndrome nefrótico y pérdida progresiva de la función renal. Con la epidemia mundial de obesidad, se produjo un aumento progresivo de la GRO del 0.2% entre 1986 y 1990 al 2% entre 1996 y 2000, y se ha convertido en un tema emergente en el ámbito de la nefrología [15]. Etiología y patogénesis de la ERC-AO La obesidad se caracteriza por un exceso de desequilibrios hormonales adipocíticos (adipoquinas), desregulación del sistema de equilibrio energético y desequilibrios en la homeostasis metabólica [12]. Hay dos tipos de tejido adiposo presentes en los humanos: tejido adiposo blanco (WAT) y tejido adiposo marrón (BAT) [17-19]. El depósito de grasa ectópica primariamente ocurre en lugares donde no se almacena fisiológicamente, como el hígado, el páncreas, el corazón y el músculo esquelético; secundariamente hay un cambio en la distribución del tejido adiposo visceral con almacenamiento de tejido adiposo en los espacios intraperitoneal y retroperitoneal; luego se presenta la desregulación inflamatoria y de adipoquinas; y por último la resistencia a la insulina [20]. Tejido adiposo blanco (WAT) El tejido adiposo blanco (WAT) se caracteriza por ser un tejido blanco o amarillo con menor vascularización e inervación que el tejido marrón. Las células grasas tienen un tamaño que oscila entre 20 y 200 µm y contienen una única vacuola lipídica (uniloculares). En dicha vacuola se almacenan lípidos para su uso cuando hay demanda energética. De la totalidad de los lípidos que abarca la vacuola lipídica del adipocito blanco, del 90 al 99% son triacilgliceroles. El tejido adiposo blanco genera una gran cantidad de adipocinas y lipocinas. Las adipocinas son péptidos que actúan como hormonas o mensajeros que regulan el metabolismo. El tejido adiposo blanco se localiza en el tejido omental, mesentérico, retroperitoneal, perirrenal, gonadal y pericárdico [19]. Este tejido al igual que el tejido adiposo de otros sitios, está compuesto por una variedad de células que incluyen macrófagos, neutrófilos, células T CD4 y CD8, células B, neutrófilos, mastocitos, células T reguladoras y células T asesinas naturales (NK) [21, 22]. El tejido adiposo es responsable de la secreción de muchas moléculas de señalización, incluidas adipocinas, hormonas, citocinas y factores de crecimiento, como leptina, adiponectina, resistina, factor de necrosis tumoral-α (TNF-α), interleucina 6 (IL-6), monocito, proteína quimioatrayente-1 (MCP-1), factor de crecimiento transformante-β (TGF-β) y angiotensina II [23]. Tejido adiposo marrón o pardo (BAT) La coloración marrón del tejido adiposo se debe a que está más vascularizado y tiene un alto contenido de mitocondrias, las células grasas que componen el tejido adiposo pardo son multiloculares o tienen varias vacuolas lipídicas. Estas células tienen forma poligonal y miden de 15 a 50 µm. A diferencia del tejido adiposo blanco, el tejido marrón no tiene la función de almacenar energía, sino que la disipa a través de la termogénesis. Para lograr la regulación de la temperatura corporal, el tejido adiposo pardo se localiza en sitios superficiales y profundos [18]. Clasificación de la ERC-AO Se ha establecido que la obesidad es una enfermedad con un comportamiento pro inflamatorio crónico con múltiples comorbilidades asociadas [19]. El tejido adiposo como se describió previamente funciona como un órgano con actividad endocrina y esta infiltrado por diferentes poblaciones celulares que incluyen macrófagos y otras células con actividad inmune como linfocitos T, B y células dendríticas [19]. La mayor parte de la grasa corporal total, se considera como un sistema de órganos endocrinos, la perturbación de este tejido tiene como resultado una respuesta patológica al balance calórico positivo en individuos susceptibles que directa e indirectamente contribuye a la enfermedad cardiovascular y metabólica, se tiene conocimiento de tres principales mecanismos de disfunción del tejido adiposo “adiposopatía” [20]. Estos mecanismos incluyen alteraciones hemodinámicas, metabólicas e inflamatorias, lo que es la base de la clasificación de la ERC-AO propuesta en esta revisión (Tabla 1). ERC-AO tipo 1 La obesidad produce un daño renal de forma directa a través de alteraciones hemodinámicas, inflamatorias, y desregulación de factores de crecimiento y adipocitoquinas, además de aumento de leptina y disminución de adiponectina, aun cuando la función renal y las pruebas convencionales sean normales [16]. La obesidad desencadena una serie de eventos, que incluyen resistencia a la insulina, intolerancia a la glucosa, hiperlipidemia, aterosclerosis e hipertensión, todos los cuales están asociados con un mayor riesgo cardiovascular [4, 16] (Figura 1). La obesidad conduce a un incremento en la reabsorción tubular de sodio, alterando la natriuresis y provocando una expansión de volumen extracelular debido a la activación del sistema nervioso simpático (SNS) y el sistema renina-angiotensina-aldosterona (SRAA)(16). El aumento en la reabsorción tubular de sodio y la consiguiente expansión de volumen extracelular es un evento central en el desarrollo de HTA en la obesidad [4, 16]. Algunos estudios sugieren que se produce un aumento de la reabsorción de sodio en algunos segmentos además del túbulo proximal, posiblemente en el asa de Henle. Además, hay un aumento del flujo sanguíneo renal, la tasa de filtración glomerular (TFG) y la fracción de filtración [16]. La hiperfiltración glomerular, asociada con el aumento de la presión arterial y otras alteraciones metabólicas como la resistencia a la insulina y la DM, finalmente resultan en daño renal y disminución del filtrado glomerular [16]. Por otro lado, la activación del SNS también contribuye a la hipertensión relacionada con la obesidad [4]. Hay evidencia de que la denervación renal reduce la retención de sodio y la hipertensión en la obesidad, lo que sugiere que la activación del SNS inducida por la obesidad aumenta la presión arterial principalmente debido al estímulo de retención de sodio, más que a la vasoconstricción [16]. Los mecanismos que conducen a la activación del SNS en la obesidad aún no se conocen por completo, pero se han propuesto varios factores como desencadenantes de este estímulo, entre ellos la hiperinsulinemia, la hiperleptinemia, el aumento de los niveles de ácidos grasos, los niveles de angiotensina II y las alteraciones del reflejo barorreceptor. El aumento de los niveles de leptina está asociado a la activación del SNS y su efecto sobre el aumento de los niveles de presión arterial incluye también la inhibición de la síntesis de óxido nítrico (potente vasodilatador) [16, 24, 25].También se ha descrito un aumento de la producción de endotelina-1 en sujetos obesos, lo que contribuye aún más a la elevación de los niveles de presión arterial y, en consecuencia, a la disfunción renal. Estudios recientes han demostrado que la endotelina-1 está aumentada en pacientes con hipertensión intradiálisis, lo que sugiere que esta sustancia juega un papel clave en la génesis de la hipertensión en pacientes con ERC y posiblemente esté asociada con la hipertensión en pacientes obesos [16, 25]. Por lo anterior, las alteraciones hemodinámicas en los pacientes con obesidad conllevan a progresión de la ERC e incremento del riesgo cardiovascular derivado del desarrollo de enfermedades adicionales como la HTA, potencialmente estos cambios son reversibles con el control de la obesidad. ERC-AO Tipo 2 Mantener el estado de obesidad más allá de los efectos renales funcionales produce cambios estructurales irreversibles a nivel glomerular [25]. El estudio de pacientes con ERC y obesidad ha permitido identificar la presencia de enfermedad glomerular asociada a la obesidad, denominada glomerulopatía relacionada con la obesidad (GRO). En esta condición la hipertrofia glomerular parece ser la lesión inicial que estimula el borramiento de los podocitos y desencadena la respuesta inflamatoria local [25, 26]. Es relevante mencionar que las señales profibrogénicas inducen la formación de depósitos en la matriz extracelular de las nefronas, que conduce al engrosamiento de la membrana basal glomeruloesclerosis y fibrosis tubulointersticial [26]. Dentro del curso patogénico de la enfermedad la expansión de la superficie glomerular conduce a que los podocitos sean incapaces de cubrirla, esto lleva a disfunción y borramiento de los mismos, generando ruptura de la barrera de filtración glomerular con sobrecarga de las células restantes, lo que finalmente conduce a hiperfiltración y proteinuria [25, 26]. No obstante, no todos los pacientes con obesidad o IMC aumentado desarrollan ERC, lo cual sugiere que el incremento del IMC por sí solo no genera aumento en la incidencia o progresión de la ERC, ameritando alteraciones metabólicas adicionales. En los siguientes apartados se describen algunas de estas vías fisiopatológicas comunes a todos los tipos de ERC-AO. ERC-AO Tipo 3 La obesidad produce daño renal de forma secundaria ya que aumenta el riesgo de diabetes mellitus, hipertensión y daño cardiovascular, estas patologías causan enfermedad renal diabética (ERD), nefroangioesclerosis, y glomerulopatía asociada a hipertensión pulmonar e insuficiencia cardíaca. La mortalidad no solo se ve afectada por la presencia de la obesidad sino por la presencia de diabetes tipo 2, hipertensión arterial, hipertensión pulmonar e insuficiencia cardíaca. Los peores resultados en supervivencia lo padecen los pacientes con falla cardíaca, obesidad e insuficiencia renal. ERC-AO Tipo 4 En pacientes en hemodiálisis los niveles más elevados de adiponectina se asocian paradójicamente con tres veces más riesgo de muerte [24]. La obesidad se asocia a niveles muy bajos adiponectina por lo que la obesidad en el grupo poblacional que se realiza hemodiálisis es un fuerte factor protector con mejores resultados de supervivencia a 3 años comparados con pacientes con índice de masa corporal normal o baja. Mecanismos fisiopatológicos comunes en la ERC-AO Lipotoxicidad derivada del tejido adiposo En pacientes obesos el exceso de energía conduce a un microambiente sometido a estrés crónico, lo cual resulta en hipertrofia del tejido adiposo hasta que los adipocitos alcanzan su límite de crecimiento [25]. En ese momento, el exceso de especies toxicas lipídicas se acumula ectópicamente en diferentes órganos, induciendo un efecto nocivo conocido como lipotoxicidad; especialmente a nivel renal [27]. La lipotoxicidad se asocia a cambios estructurales y funcionales de las células mesangiales, podocitos y células tubulares proximales [28]. En los podocitos, esto interferiría con la vía de la insulina, crítica para la supervivencia y el mantenimiento de la estructura de los podocitos, lo que conduciría a la apoptosis de los podocitos e induciría una respuesta hipertrófica compensatoria en los podocitos restantes [25]. En el riñón, los depósitos de lípidos ectópicos contribuyen tanto a la inflamación local como al estrés oxidativo [27]. En modelos de ERD, la dislipidemia puede favorecer la acumulación de lípidos ectópicos e intermediarios lipídicos, no solo en el riñón sino también en tejidos extrarrenales como hígado, páncreas y corazón [27]. La acumulación de lípidos en el parénquima renal, genera daño en varias poblaciones celulares, incluídos podocitos, células epiteliales tubulares proximales y el tejido tubulointersticial a través de distintos mecanismos descritos en las siguientes apartados, pudiendo general compromiso a largo plazo de la función renal [27]. El tejido adiposo es una fuente importante de producción de diferentes factores proteicos activos, conocidos como adipocitocinas, las cuales participan en diferentes procesos metabólicos. Alteraciones en la secreción y señalización de moléculas derivadas del tejido adiposo durante la obesidad en gran medida puede mediar en la patogenia de los trastornos metabólicos [25]. A continuaciones se describe el rol de las adipocinas en la patogenia de la ERC y obesidad. Adiponectina La adiponectina es una proteína secretada principalmente por los adipocitos WAT, las principales funciones biológicas de la adiponectina incluyen una mayor biosíntesis de ácidos grasos y la inhibición de la gluconeogénesis hepática [17]. Es probablemente la adipocina secretada más abundantemente, forma alrededor del 0.05 % de las proteínas séricas y mide de 3 a 30 mg/ml en humanos, para su activación utiliza dos isoformas del receptor (AdipoR1 y AdipoR2) son receptores de siete transmembranas y tienen una homología del 66.7 % en su estructura [17]. Sin embargo, AdipoR1 y AdipoR2 son estructural y funcionalmente distintos de los receptores acoplados a proteína G porque su terminal N es intracelular, mientras que el terminal C es extracelular [29, 30]. La señalización de adiponectina se basa principalmente en interacciones de tipo receptor-ligando, en las que la adiponectina se une a sus receptores afines e inicia la activación de varias cascadas de señalización intracelular a través de las vías AMPK, mTOR, NF-κB, STAT3 y JNK [17]. La adiponectina inicia la activación de la señalización de AMPK mediada por la proteína adaptadora APPL1, que se une al dominio intracelular de AdipoR. Eso produce la activación de la biosíntesis de moléculas, otras proteínas reguladoras e importantes factores de transcripción. AMPK es un regulador que participa principalmente en la proliferación celular [17]. Hay dos tipos de macrófagos, M1 participan en la estimulación de los factores pro inflamatorios e induce la resistencia a la insulina y M2 bloquean una respuesta inflamatoria y promueve el metabolismo oxidativo; En los macrófagos, la adiponectina promueve la diferenciación celular de monocitos a macrófagos M2 y suprime su diferenciación a macrófagos M1, lo que muestra efectos pro inflamatorios y antiinflamatorios. Además, también activa los factores antiinflamatorios IL-10 pero reduce las citoquinas pro inflamatorias como IFN-γ, IL-6 y TNF-α en los macrófagos humanos [17]. Los pacientes con ERC muestran niveles elevados de proteína C reactiva (PCR), IL-6 y TNF-α y tienen una activación aberrante de receptor tipo toll (TLR)-4 [25]; en un estudio realizado en el año 2005 en 29 pacientes con ERC no diabéticos en etapa 5 y 14 controles sanos, se identificó que los pacientes con ERC tenían una expresión elevada del gen y la proteína TLR4, la estimulación de TLR-4 in vitro indujo la activación de TNF-α y NF-κB en células C2C12. Esto sugiere indirectamente que la activación de TLR-4 podría promover la inflamación muscular de los pacientes con ERC [31]. Los niveles de adiponectina se consideran predictivos de ERC, dado que estos se encuentran aumentados en pacientes con etapa pre diálisis [17, 29, 32]. Adicionalmente, en un estudio prospectivo realizado en el año 2008 en pacientes con ERC primaria no diabética identificó niveles elevados de adiponectina como un predictor novedoso de progresión de la ERC en hombres [33]. En estudios realizados en animales (ratones) muestran que la deficiencia de adiponectina se relaciona con varias alteraciones histológicas, incluida la fusión segmentaria procesos podocitarios, albuminuria y aumento del estrés oxidativo en los riñones [34]. Por otro lado, en pacientes obesos la producción de adiponectina se encuentra disminuida por lo que se cree que puede generar una función protectora sobre el riñón [29]. No obstante, paradójicamente, algunos estudios muestran que los pacientes con ERC y enfermedad renal crónica en diálisis (ERCT) tienen altos niveles de adipocinas, las explicaciones a esta situación son controversiales, se ha planteado podrían corresponder a un mecanismo compensatorio, otras consideraciones sugieren una disminución de la sensibilidad a la adiponectina o una reducción en el aclaramiento de la misma [35]. Leptina En pacientes con ERC independiente de la presencia de obesidad o no, se asocian a niveles elevados de leptina sérica. La leptina es una proteína de 167 aminoácidos, con una masa molecular de aproximadamente 16 kDa que está codificada por el gen LEP [23] secretada principalmente por los adipocitos, es una adipocina pleiotrópica. La leptina circulante llega a los órganos diana, donde se une a receptores específicos (conocidos como ObR, LR o LEPR), se conocen cinco isoformas del receptor de leptina en humanos (ObRa, ObRb, ObRc, ObRd y ObRe), de estas solo la isoforma ObRb (isoforma larga) se considera un receptor completamente activo, ya que es capaz de transducir completamente una señal de activación en la célula. Esta isoforma se encuentra altamente expresada en el sistema nervioso central (SNC), especialmente en el hipotálamo, donde participa en la regulación de la actividad secretora de este órgano. Los efectos de la leptina están mediados por cinco vías principales de señalización. Estas vías incluyen las vías de señalización JAK-STAT, PI3K, MAPK, AMPK y mTOR [23]. Por esta razón la principal función fisiológica de la leptina es transmitir información al hipotálamo sobre la cantidad de energía almacenada, como la masa de tejido adiposo, e influir en el gasto de energía al reducir el apetito. Regula el metabolismo energético, tiene efecto sobre la ingesta de alimentos, procesos de coagulación, angiogénesis, funciones relacionadas con la insulina y la remodelación vascular, además funciona como un pro inflamatorio molecular [36]. La leptina tiene efectos sobre el apetito y se ha demostrado que la hiperleptinemia contribuye a la hipertensión asociada a la obesidad por sobre activación del sistema nervioso simpático [37]. En cuanto al curso de la ERC, la leptina puede modular diferentes vías de señalización en el riñón, debido a que las células endoteliales glomerulares y mesangiales expresan abundantes receptores de leptina [25]. La leptina inducirá un incremento en la expresión de genes profibróticos, como TGF-β1 y citocinas pro inflamatorias [25]. El aumento en la expresión de TGF-β1, también contribuirá al desarrollarlo de la fibrosis renal, al unirse a receptores específicos a nivel renal, estimulara la expresión de factores profibróticos en un ciclo de retroalimentación positiva. Además, TGF-β1 es un potente iniciador de proliferación de células mesangiales renales [25]. Debido a su tamaño relativamente pequeño, la leptina atraviesa libremente el filtro glomerular de los riñones y luego se reabsorbe en la parte proximal de los túbulos contorneados [23]. Por lo que el estado elevado de leptina puede indicar una función renal deficiente [36]. Promueve la inflamación y trastorno de los lípidos, que contribuyen al riesgo de ERC [36]; se considera como “toxina urémica”, estando implicada tanto en la progresión de la enfermedad renal a través de efectos pro-hipertensivos y profibróticos, como en el desarrollo de complicaciones relacionadas con la ERC (inflamación crónica, pérdida de proteínas) [38]. Como se mencionó previamente, la leptina estimula la proliferación de células endoteliales glomerulares renales y aumenta la expresión de TGF-β1, un mediador clave de la hidrogénesis en estas células, el aumento de los niveles de leptina también contribuye al aumento de la expresión de colágeno tipo IV en el riñón, induce la proliferación de células mesangiales glomerulares mediante la activación de la vía PI3K, la hipertrofia de las células mesangiales aumenta la cantidad de proteína filtrada y albúmina que llega a las células del túbulo proximal y, como resultado, activa las vías inflamatorias y la fibrosis [23]. Puede presentarse un aumento en la síntesis del receptor TGFβ-1 secretado por las células endoteliales, este actúa de manera parácrina sobre el mesangio uniéndose a su receptor y activando la síntesis de proteínas de la matriz extracelular (ECM), incluyendo colágeno, fibronectina, tenazina y proteoglicanos; consiguientemente, un aumento en el nivel de TGFβ-1 conduce a la acumulación de MEC y, en consecuencia, a fibrosis glomerular y glomeruloesclerosis. En los podocitos, la leptina contribuye a la disminución de la expresión de las proteínas responsables de la filtración glomerular adecuada, incluidas la podocina, la nefrina, la podoplanina y la podocalixina. En las células del túbulo contorneado proximal (PTC), la leptina reduce la actividad metabólica de las células al activar la vía de señalización de mTOR [23]. Por otro lado, la leptina inhibe el apetito y aumenta el gasto de energía conduciendo a anorexia y desnutrición en pacientes con ERC, particularmente en casos de hemodiálisis de mantenimiento [36]. Por ende, una elevación de la leptina no solo nos indicaría daño renal, sino que además nos indica mayor progresión de complicaciones secundarias [39]. La obesidad aumenta la carga sobre los riñones y es un factor de riesgo de lesión renal, además de contribuir en los trastornos metabólicos asociados. Por lo que, teniendo en cuenta los efectos inhibitorios de la leptina sobre la obesidad, se puede considerar que puede proteger contra la lesión renal [39, 40]. Un estudio experimental publicado en el año 2017 demostró que la leptina disminuyó la ingesta calórica y los niveles de glucosa en ratas diabéticas [41], ese mismo año se publicó un estudio retrospectivo donde demostraron que la metreleptina, una metionil leptina humana recombinante, reduce el peso corporal y la dosis diaria de insulina en la diabetes mellitus tipo 1 [42]. La metreleptina ejerce efectos terapéuticos en la lipodistrofia [43], lo que indica que es probable que la leptina se aplique en los trastornos metabólicos [36]. Otras adipocinas Las principales adipocinas corresponden a la adiponectina y leptina como se ha descrito previamente. Además de estas, se distinguen la actividad de la visfatina y resistina, las cuales muestran propiedades pro-inflamatorias y efectos aterogénicos [25]. La visfatina estimula la expresión de TGF-β1, inhibidor del activador del plasminógeno-1 (PAI-1) y colágeno tipo I, los cuales han demostrado un rol importante como agentes profibróticos. Por otro lado, la resistina estimula la producción de las moléculas de adhesión como la molécula de adhesión intracelular 1 (ICAM-1) y la proteína de adhesión celular vascular 1 (VCAM-1) y promueve la activación del sistema renal simpático. Los niveles de estas adipocinas están marcadamente elevados en la obesidad y ERC correlacionándose con parámetros proinflamatorios y disminución de la tasa de filtración glomerular (TFG) [25, 37]. Durante el curso de la obesidad se presenta una sobre activación del SRAA, el tejido adiposo también estaría involucrado en la producción o estimulación de algunos de los componentes del RAS. Por ello la sobre estimulación del SRAA en obesos, asociado a la glomerulomegalia y desregulación de la reabsorción de sodio/glucosa, generalmente conlleva a hipertensión glomerular e hiperfiltración [25]. Otra adipocina a considerar, es la actividad de la adipocina proinflamatoria lipocalina 2 (LCN2), también denominada lipocalina asociada con la gelatinasa de neutrófilo (NGAL), estudiada como biomarcador funcional tanto para la enfermedad renal aguda como ERC(25). LCN2 es conocido por su papel en la respuesta inmune innata a través de su unión a sideróforos derivados de una infección bacteriana. Sin embargo, LCN2 no es secretada únicamente por neutrófilos sino también por otros tejidos como hígado, pulmones y de interés para este artículo, a nivel renal [25]. Se han informado niveles elevados de LCN2 en suero y orina en la lesión renal, debido a una expresión aumentada de LCN2 en el túbulo distal renal y una reabsorción alterada en el túbulo proximal [44]. El tejido adiposo, también puede producir factores angiogénicos como el factor de crecimiento del endotelio vascular (VEGF). Este elemento podría inducir la formación de novo de capilares glomerulares en gran parte defectuosos dentro del riñón, lo que contribuye a la hipertrofia glomerular característica de GRO [25] (Figura 2). Conclusiones La obesidad y el sobrepeso se asocian a alteraciones hemodinámicas, estructurales e histopatológicas en el riñón, así como alteraciones metabólicas y bioquímicas que predisponen a la enfermedad renal, aun cuando la función renal y las pruebas convencionales sean normales. Por lo tanto, los efectos renales de la obesidad son estructurales y funcionales. Hay varios mecanismos actualmente descritos que involucran a la obesidad como generador de alteraciones renales. Teniendo en cuenta las bases fisiopatológicas, proponemos una clasificación de la ERC-AO basadas en 4 tipos. Abreviaturas ERC: enfermedad renal crónica. ERC-AO: enfermedad renal crónica-asociada a enfermedad. VEGF: factor de crecimiento del endotelio vascular. OR: Odds ratio. Información suplementaria Materiales suplementarios no han sido declarados. Agradecimientos No aplica. Contribuciones de los autores Jorge Rico-Fontalvo: Conceptualización, Curación de datos, Análisis formal, Adquisición de fondos, Investigación, Metodología, Administración de proyecto, Recursos, Software, Escritura – borrador original. Rodrigo Daza-Arnedo: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Tomás Rodríguez-Yanez: Metodología, validación, supervisión, redacción: Revisión y edición. Washington Osorio: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Beatriz Suarez-Romero: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Oscar Soto: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Juan Montejo-Hernandez: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. María Cardona-Blanco: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Juan Camilo Gutiérrez: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Todos los autores leyeron y aprobaron la versión final del manuscrito. Financiamiento Los autores proveyeron los gastos de la investigación. Disponibilidad de datos o materiales Los conjuntos de datos generados y analizados durante el estudio actual no están disponibles públicamente debido a la confidencialidad de los participantes, pero están disponibles a través del autor correspondiente a pedido académico razonable. Declaraciones Aprobación del comité de ética y consentimiento para participar No aplica para revisiones narrativas. Consentimiento para publicación No aplica cuando no se publican imágenes o fotografías del examen físico o radiografías/tomografías/resonancias de pacientes. Conflictos de interés Los autores reportan no tener conflictos de interés. 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Abstract Background The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. Methods Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. Results We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. Conclusions Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.