Academic literature on the topic 'Calreticulina, MPN, CRISPR-Cas9'

Abstract Introduction: Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype. Methods: We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential. Results: Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody. Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation. When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors. To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF. Conclusion: Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. Disclosures Greinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

Abstract INTRODUCTION. Calreticulin (CALR) is mutated in 20% of pts with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most frequent mutations are a 52 bp deletion (T1) and a 5 bp insertion (T2) in exon 9, that cause a recurrent frameshift resulting in novel C-terminal sequence common to all mutant CALR proteins. Recent data indicate that interaction of mutant CALR with the thrombopoietin receptor MPL contributes to the abnormal megakaryocytopoiesis (Mk) of ET/PMF (Araki M et al & Chachoua I et al, Blood 2016; Elif S et al, Blood 2018), however full characterization of mechanisms pertaining to mutant CALR remains to be pursued. METHODS. By using CRISPR/Cas9 editing, we generated CALR knock-out (KO) variants starting from cord blood (CB) CD34+ cells, K562, UT7 and HL-60 cell lines, and CALR T1 variants from K562 and UT7 cells. Stable expression of CALR wild-type (WT), T1 and T2 was obtained by viral transfection of K562-KO cells. RESULTS. In the different genome-edited cell models, KO or T1 mutation did not result in appreciable changes in proliferation rate, cell cycle and apoptosis under standard culture conditions. However, UT7 KO and T1 cells were able to grow in the absence of GM-CSF, that was otherwise necessary for maintenance of WT counterpart, indicating cytokine independence similarly imparted by deletion or T1 mutation of CALR. To evaluate impact of mutated CALR on Mk commitment, we induced parental, KO and T1 K562 cells with phorbol-myristate acetate (PMA); at day 3 after induction, 60% and 48% of KO and T1 cells, respectively, expressed CD41/CD61 compared to 24% of parental cells (p<0.01). Similar findings were noted in KO K562 cells that were transfected with vectors encoding the WT, T1 and T2 mutated CALR: CD41/CD61+ cells increased from 30% in parental cells to 70%, 55%, 62%, respectively, in KO cells and cells expressing T1 and T2 mutant CALR (p<0.01). We also found that a greater proportion of KO and T1 UT7 cells, maintained in either GM-CSF and TPO, spontaneously expressed CD41/CD61 at d7 of culture (50-51% and 41-49%, respectively, for KO and T1 cells with GM-CSF or TPO compared to 26-25% of parental cells; p<0.01) and underwent morphologically recognizable Mk maturation. Mk clonogenic assays were initiated in plasma clot cultures by plating CB-derived CD34+ cells with CRISPR/Cas9-induced deletion of CALR (KO cells). KO CD34+ cells generated a number of CFU-Mk colonies that was 10-fold higher than untouched CD34+ cells (p<0.05). Overall, these data suggested that deletion of CALR results in promotion of Mk differentiation in cell lines and primary cells thereby mimicking the effects of CALR T1. Array phosphoproteomic assay in K562 KO cells showed phosphorylation levels of p38γ, p70S6k mTOR, Erk1 and Erk2 from 0.5 to >2-fold higher compared to parental cells. Then, we induced KO and T1 K562 cells to Mk differentiation in the presence of JAK2, mTOR and Erk1/2 -inhibitors; only mTOR and Erk1/2 -inhibitors significantly (P< 0.05) reduced Mk differentiation of T1 and KO cells by respectively 5- and 4- fold with mTOR inhibitor and 5- and 4.5- fold with Erk1/2 inhibitor compared to parental cells, that were largely insensitive to inhibitors (Figure). Isolated megakaryocytes from CALR-mutant patients were found to present abnormally increased release of Ca2+ from endoplasmic reticulum (ER) compared to control cells (Pietra D et al. Leukemia, 2016). We similarly found that both KO and T1 K562 cells presented impaired capability in restoring calcium homeostasis compared to parental cells under treatment with ionophores thapsigargin and ionomycin. Finally, we comparatively assessed MPO expression by flow cytometric analysis in parental and CALR KO HL-60 cell lines; increased myeloperoxidase (MPO) degradation was previously reported in MPN pts expressing CALR mutation (Theocharides A et al., Blood, 2016). We found significant reduction of MPO levels (9-fold lower, p<0.01) in KO compared to parental cells, while MPO mRNA levels were not affected by CALR KO, suggesting similarly enhanced degradation of MPO in cells with targeted deletion of CALR. CONCLUSIONS. Overall, our data suggest that the novel C-terminus of CALR originating from exon 9 mutations loses some physiologically functions that are phenocopied by absence of CALR protein in cells made KO for the gene in a MPL-independent context. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

【Introduction】 Although Ruxolitinib (Rux), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients. Currently, Rux-resistance is a major problem and contributes to poor prognosis of MPN patients. Mechanisms of Rux-resistance in MPN cells with JAK2V617F mutations have already been reported. On the other hand, MPN cells with CALR mutations resistant to Rux-treatment have not been fully characterized yet. In this study, we have clarified a mechanism of Rux-resistance in MPN tumor cells with CALR mutations in both cell lines and clinical samples. 【Materials and Methods】To establish human cell lines with CALR mutations, we have introduced deletion of 46-nucleotides generating the CALR frame-shift mutation (del46), one of the type1-like mutations, to UT-7/EPO by the CRISPR/Cas9 system. Then, we introduced exogenous MPL to UT-7/EPO with the CALR mutations by transfection. We confirmed that UT-7/EPO with CALR frame-shift mutations (CALR-fs) and exogenous MPL (exMPL) activate STAT5 and showed cytokine-independent cell growth. To establish Rux-resistant cell lines, we have cultured these cell lines with low-dose Rux (0.2μM), and gradually increased the concentrations of Rux by 0.1μM every week. Cells were considered as resistant when the half-maximal inhibitory concentrations (IC50) of the cells was double the IC50 of the parental cells. We have successfully established Rux-resistant cells that proliferated in the presence of Rux at 0.8μM. Then, we characterized the Rux-resistant cells with CALR-fs/exMPL. Next, reversibility of Rux-resistance, the Rux-resistant cells was examined. To examine whether Rux-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted Rux-resistant cells as well as Rux-sensitive cells into immunocompromised mice and treated the animals with Rux. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors were excised and weighted. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We found that Rux-resistant cells had overexpressions of both MPL and JAK2, and increased phosphorylations of both JAK2 and STAT5. We found that high levels of MPL transcripts in the resistant cells. Cycloheximide treatment assay showed that mature MPL proteins were more stable in Rux-resistant cells. Then, we examined mechanisms of stability of mature MPL proteins in Rux-resistant cells. Our data suggested that the proteasome/system degrading mature MPL was attenuated in Rux-resistant cells. When Rux-resistant cells were cultured in the absence of Rux, Rux-resistance was reversed with reduction of mature MPL and JAK2. In vivo assay using immunocompromised mice showed the Rux resistant cells were not sufficiently suppressed by Rux-treatment. Since our data using the cell lines suggested that high MPL expression causes resistance to Rux in MPN cells with CALR mutations, we examined levels of MPL proteins in bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. Immunohistochemical staining of MPL showed that the megakaryocytes of MPN patients with high-risk and/or resistant to ruxolitinib treatment expressed higher MPL. 【Discussion】 MPL overexpression is one of mechanisms of MPN cells with CALR mutations to gain resistance to Rux. This mechanism may be a key element to overcome Rux-resistance in this fatal disease. Disclosures Komatsu: Novartis K.K: Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau.

Abstract Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis. We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values &lt;0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings. Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis. We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a &gt;50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells. To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells. We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p&lt;.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03). We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p&lt;0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells. Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN. In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN. AEM and JSJ contributed equally. Figure 1 Figure 1. Disclosures Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

Introduction. Frameshift mutations in exon 9 of calreticulin (CALR) gene represent the second most frequent alteration in MPN. More than 30 types of CALR mutations are described; the most frequent are a 52bp deletion (Type1, T1) and a 5bp insertion (T2). All mutations cause a recurrent frameshift that originates a novel C-terminal peptide lacking the KDEL Endoplasmic Reticulum-retention sequence. Recent data described the interaction of mutated CALR (CALRm) with the thrombopoietin receptor MPL and the resulting activation of JAK/STATs as well MAPK and PI3K pathways (Araki M et al & Chachoua I et al, Blood 2016; Elf S et al, Blood 2018). However, full characterization of the molecular mechanisms linking CALRm with MPN remains to be fully accomplished. Methods. By using CRISPR/Cas9 technology we generated CALR knock out (KO) and T1 variants from the GM-CSF cytokine dependent UT7 cell line; primary CD34+ cells from CALRm pts were also analyzed. Results. Introduction of CALR T1 mutation or generation of KO status did not appreciably modified the proliferation rate, cell cycle and apoptosis of UT7 cells under standard conditions. However, UT7 T1 and KO cells were similarly able to grow in the absence of GM-CSF, otherwise necessary for WT cells; in these cytokine-depleted conditions, apoptosis was significantly increased in WT cells compared both T1 and KO (4- and 8-fold higher; p&lt;0.01) suggesting acquisition of cytokine independency. Exploring the mechanisms leading to cytokine independence we found that signal transducer and activation of transcription 3 (STAT3) was the main constitutively phosphorylated STAT in T1 and KO cells. Among a panel of STAT3 activation candidates assessed by RT-PCR, we found that mRNA levels of interleukin 6 (IL6), which induces STAT3 phosphorylation and activation through the glycoprotein 130/Janus kinase (GP130/JAK) pathway, were significantly increased in T1 and KO compared to parental cells (2- and 3.5-fold higher; p&lt;0.01). The levels of IL6 released in cell culture medium were also significantly increased (1.5- and 2-fold higher; p&lt;0.05). Blocking GP130 with SC144 or IL6 receptor (IL6R) with Tocilizumab (TCZ), in cytokine-depleted conditions, led to cell growth arrest in T1 and KO with no effects on WT cells. SC144 treatment induced a marked dephosphorylation of STAT3 in T1 and KO cells. Also, the membrane expression of GP130 and IL6R resulted higher in KO and T1 compared to WT cells, whilst intracellular content was similar. Co-immunoprecipitation showed that wild type calreticulin, unlike the mutated T1, formed complexes with GP130 and IL6R suggesting that CALRm lost the capacity to negatively regulate their membrane exposure resulting in downstream increased IL6 synthesis and release. The role of IL6 pathway in CALRm patients was evaluated by assessing megakaryocyte (Mk) colony generation in plasma clot cultures of CD34+ cells in the absence/presence of IL6, as part of cytokine cocktail optimally supporting CFU-Mk growth. We found that CFU-Mk generation by CALRm CD34+ cells was largely independent of exogenously added IL6, unlike control CFU-MK whose number was halved in the absence of IL6 (p&lt;0.05). Moreover, inhibition of GP130 and IL6R by SC144 and TCZ, respectively, significantly reduced CFU-Mk by CALRm CD34+ cells (2.9- and 3.2-fold; p&lt;0.05), with no effect in control cultures. As anticipated, JAK inhibitors ruxolitinib and momelotinib reduced CFU-Mk formation by CALRm CD34+ cells (2- and 3.2-fold, respectively; p&lt;0.01), particularly evident in exogenous IL6-free conditions (5.8- and 4.8-fold, respectively; p&lt;0.01). The combination of either ruxolitinib or momelotinib with TCZ on CFU-Mk formation was also assessed. Both combinations proved synergistic activity in reducing CFU-Mk from CALRm CD34+ cells (4.3- and 5-fold vs control cultures, respectively; p&lt;0.01; combination compared to single agent, 2.8- fold vs ruxolitinib, 4.5-fold vs momelotinib and 4-fold vs TCZ, p&lt;0.05 for all). Conclusions. Overall, our data suggest a novel model of action of mutant CALR resulting from the loss of its capability to complex GP130 and IL6R and regulate their cell membrane expression. Augmented expression of IL6 receptor complex on CALRm CD34+ cells on turn results in deregulated IL6-dependent signaling and augmented megakaryocyte generation. Blockade of IL-6/GP130/JAK signaling pathway might represent a new therapeutic target in pts with CALR mutation. Disclosures Vannucchi: Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Somatic mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, are found in up to 40% of patients with myeloproliferative neoplasms (MPN). All pathologic CALR mutations are out-of-frame insertion and/or deletions (indels) in exon 9, generating a 1 base-pair (bp) frame shift and a common mutant-specific C-terminus, with the most common mutation being a 52 bp deletion (del52). The observation that CALR mutations are mutually exclusive with other MPN-initiating mutations such as JAK2V617F suggests a key pathogenic role for mutant CALR. To determine if mutant CALR alone is sufficient to induce MPN we began by over-expressing CALR-del52 in a retroviral bone marrow transplant (BMT) mouse model. We found that CALR-del52-expressing mice develop thrombocytosis and megakaryocytic hyperplasia, recapitulating the megakaryocyte-specific phenotype of CALR-mutant MPN patients. These findings suggest that the thrombopoietin receptor, MPL plays a key role in the pathogenesis of mutant CALR-driven MPN. To evaluate the role of MPL in mutant CALR driven oncogenesis, we over-expressed CALR-del52 in interleukin-3 (IL-3)-dependent Ba/F3 hematopoietic cells. We found that CALR-del52 over-expression results in transformation to IL3-independent growth only in Ba/F3 cells co-expressing MPL, but not in parental Ba/F3 cells or Ba/F3 cells co-expressing the EPO receptor (EPOR) or the G-CSF receptor (GCSFR). We found similar results in human cytokine-dependent UT-7 cells. We also introduced +1 frameshift mutations into the endogenous Calr locus in Ba/F3-MPL cells using CRISPR/Cas9 gene editing and successfully engendered IL-3 independent growth, indicating that endogenous levels of mutant Calr expression are sufficient for transformation. Together, these data indicate that MPL is specifically required for the transforming capacity of mutant CALR. Using RNA-sequencing followed by gene set enrichment analysis (GSEA), we confirmed that mutant CALR transformed Ba/F3-MPL cells display strong enrichment of Stat5 and Stat3 gene expression signatures. Concordantly, we also saw differential phosphorylation of Stat5 and Stat3 in these cells. Furthermore, we found that the IL-3 independent proliferation of mutant CALR expressing Ba/F3-MPL cells is decreased upon shRNA-mediated knockdown of Jak2, and that differential activation of Stat5 and Stat3 is abrogated by the JAK2 inhibitor, ruxolitinib. Together, these data demonstrate that mutant CALR signals through the JAK/STAT axis downstream of MPL. We next sought to define the specific domains within mutant CALR required for oncogenic transformation. We found that neither expression of the mutant C-terminus alone nor expression of CALR lacking the C-terminus leads to cytokine-independent growth, suggesting that the novel C-terminus is necessary (but not sufficient) for transformation. We therefore generated an extensive series of truncation, domain deletion and point mutations within the C-terminus and assessed their respective transforming capabilities. Surprisingly, we found that the oncogenic activity of mutant CALR is not encoded within a specific sequence or domain of the mutant C-terminus. Rather, we found that the positive electrostatic charge of the mutant C-terminus is critical for its transforming capacity. Mutagenizing all 18 lysine/arginine residues (positively charged) within the C-terminus to a neutral glycine residue abrogates CALR-del52 transformation activity. In contrast, mutagenizing the 18 non-lysine/arginine residues within the C-terminus to glycine does not affect transforming activity, a remarkable finding considering that, in this mutant, 50% of the amino acids have been modified. Finally, using co-immunoprecipitation assays we found that mutant CALR, but not wild-type CALR, physically interacts with MPL, and that neither the mutant C-terminus alone nor mutant CALR lacking the C-terminus can bind to MPL. This suggests that the tertiary structure of mutant CALR is required for binding to MPL. Moreover, we found that the ability of our engineered CALR mutants to bind MPL perfectly correlates with their ability to mediate transformation, suggesting that the interaction with MPL is critical for mutant CALR-mediated transformation. Together, our findings elucidate a novel mechanism of pathogenesis in MPN and provide insights into how CALR mutations drive the development of MPN. Disclosures: No relevant conflicts of interest to declare.

Abstract In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation. References 1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. N Engl J Med. 2013 Dec 10;369(25):2379-90. 2. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. N Engl J Med. 2013 Dec 10;369(25):2391-405. 3. Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Blood. 2015 Dec 14;10.1182/blood-2015-11-681932. 4. Marty C, Pecquet C, Nivarthi H, Elkhoury M, Chachoua I, Tulliez M, et al. Blood. 2015 Nov 25;10.1182/blood-2015-11-679571. 5. Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C, et al. Leukemia. 2016 Feb 29;10.1038/leu.2016.47. Disclosures Constantinescu: Teva: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice. 3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs. Conclusion We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. Disclosures Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

AbstractCalreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Chronic myeloproliferative neoplasms (MPN) originate from hematopoietic stem cell (HSC) and include polycythemia vera, essential thrombocythemia and primary myelofibrosis. They are characterized by mutations in JAK2, MPL and CALR. This project focused on the most recently described driver mutations in Calreticulin , whose functions and downstream targets are still largely unknown. There are several types of CALR mutations, the most frequent are a 52 bp (Type1) deletion and a 5 bp (Type2) insertion in exon 9. All described abnormalities cause a frameshift with generation of a novel C-terminal domain with a common novel sequence of 36 aminoacidic. In order to create a novel tool allowing mechanistic analysis of mutated CALR in a hematopoietic setting, I used CRISPR/Cas9 technology to perform targeted site-specific genome editing in K562 and UT7 cell line. I was successful in generating CRISPR/Cas9 K562 and UT7 CALR Knock-Out (KO) and CALR mutated with 52 bd delection (T1), that is an unique model where changes are to be entirely ascribed to CALR mutation and are not affected by endogenous CALR. Further characterization of K562 and UT7 CALR KO and CALR T1 showed no significant abnormalities in proliferation and cell cycle compartments compared to parental cells, while mutant cells had increased resistance to apoptosis. I showed that mutant CALR protein remained largely in the cytosol and it had a lower stability compared to wild-type counterpart. Since CALR mutations are restricted to MPN subtypes displaying aberrant megakaryopoiesis I also evaluated the effects of the mutations on the megakaryocytic commitment by culturing K562 with 10 nM PMA, a known inducer of megakaryocyte differentiation. I found that both CALR KO and T1 cell lines showed accelerated and enhanced expression of CD41/CD61 differentiation markers compared to CALR wild-type cells; this was also confirmed in K562 KO cells stably expressing CALR Type1 and Type2 by lentiviral transduction and in UT7 CALR KO and T1 culturing with PMA. Moreover clonogenic assay with CRISPR/Cas9 genome edited CD34+ cells showed that the knock-out of the protein resulted in promotion of megakaryocytopoiesis, mimicking the effects of CALR mutations. To reinforce these observations, the studies with inhibitor drugs in K562 CALR KO, CALR T1 and in CALR KO CD34+ cells, confirmed that PI3K and Erk pathways are involved in calr-mediated Mk commitment. These data were in line with results obtained with the phosphoproteomic assay in K562 CALR T1 and KO cells. Finally we assessed MPO expression in our cell models and we found that K562 CALR KO cells had a reduced expression of MPO, mimicking the observation in CALR T1 cells. Further, following the re-expression of CALR WT in CALR KO cells, we observed that MPO expression was restored, demonstrating that both mutated CALR and the absence of CALR (KO cells) lead to MPO degradation. Overall, these data indicate that the generated CALR-mutated and KO models represent useful tools to study the pathogenic and phisiologic role of CALR in MPN and could be useful to develop new diagnostic and therapeutic strategies for future clinical applications.