ALBERT

Description: Key Points Jak2 deletion in PLTs and MKs leads to thrombocytosis due to dysregulated TPO turnover. Jak2 loss in PLTs/MKs induces non-autonomous expansion of stem/progenitors, and specifically of MK-primed hematopoietic stem cells (HSCs).

Description: Among BCR-ABL-negative myeloproliferative neoplasms (MPN), primary myelofibrosis (PMF) and post PV/ET myelofibrosis (MF) are associated with the highest degree of morbidity and mortality, including progressive bone marrow (BM) fibrosis and resultant BM failure. Although the JAK inhibitor ruxolitinib is now approved for the treatment of MF-associated splenomegaly and systemic symptoms, JAK inhibitor therapy does not reduce the proportion of JAK2-mutant cells in MPN patients. The limited ability of JAK inhibition to induce molecular or clinicopathologic responses in the majority of MPN patients underscores the need for the development of more effective therapies for JAK kinase-dependent malignancies. Recent studies have shown that the lysine-specific histone demethylase, LSD1 (KDM1A), participates in the balance between proliferation and differentiation in vivo by influencing state-specific gene expression patterns. In physiologic hematopoiesis, LSD1 is essential for normal myeloid differentiation affecting the erythroid, megakaryocytic and granulocytic lineages. Small molecule inhibitors of LSD1 have shown promising results in preclinical models of acute myeloid leukemia (AML) and solid cancers and have recently entered clinical trials in AML. However, the role and requirement for LSD1 in the pathogenesis of MPNs and the therapeutic targeting of LSD1 in MPN has not been investigated. In this study, we first tested the effects of IMG-98, a potent, selective LSD1 inhibitor, in the MPLW515L-driven ET/MF mouse model. After disease was established, mice were treated with IMG-98 or vehicle for 28 days. LSD1 inhibition in mice markedly suppressed myeloproliferation reducing granulocyte counts and spleen weights compared to mice treated with vehicle thus establishing therapeutic efficacy (Fig. 1a). Pathologic analysis of BM and spleen confirmed a marked reduction in myeloproliferation as well as a reversal of extramedullary hematopoiesis (EMH). Most notably, we observed a marked reduction in reticulin fibrosis with IMG-98 treatment (Fig. 1b). We next investigated the impact of IMG-98 therapy on inflammatory cytokine signaling; in contrast to the broad anti-cytokine effects of JAK1/2 inhibition, we observed a more specific anti-cytokine effect of IMG-98, a significant reduction in the secretion of the inflammatory cytokine Cxcl5 (Fig. 1c), a key participant in pathologic inflammatory states. We then investigated the in vivo impact of IMG-98 therapy on mutant disease burden. IMG-98 therapy reduced mutant allele burden to a degree not seen with JAK1/2 inhibitor therapy: whereas 74.6% of circulating cells in mice treated with vehicle were GFP-positive cells, only 43.2% of circulating cells were GFP-positive in IMG-98-treated mice (Fig. 1d). Flow cytometry analysis of spleen and BM revealed reduced numbers of CD11b/Gr1-positive myeloid cells and CD41-positive megakaryocytes. The numbers of mutant GFP-positive myeloid cells and megakaryocytes in these tissues were also significantly reduced by IMG-98 treatment. Studies of the impact of LSD1 inhibition on MPN stem cell function and on epigenetic regulation in MPN cells will be presented in detail. In summary, the LSD1 inhibitor IMG-98 had a highly significant therapeutic effect in an established preclinical model of ET/MF. LSD1 inhibition in diseased mice reduced JAK-STAT-driven myeloproliferation, markedly reversed EMH and BM fibrosis, and reduced the mutant clone burden. These data suggest LSD1 is a valid target in MPN and that clinical studies of LSD1 inhibitor IMG-98 alone and in combination with JAK inhibitors are warranted. Figure 1. a, b) LSD1 inhibition results in reduced white blood cell counts (WBC) and platelet counts (PLT). (a), and in near-complete elimination of BM fibrosis (b). c) Profound reduction of Cxcl5 serum levels in IMG-98 treated mice compared to vehicle treated mice. d) Significantly lower mutant allele burden in the peripheral blood of IMG-98 treated mice. * P

Description: The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity. Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Koppikar: Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

Description: We have recently reported inactivation of the tyrosine phosphatase PTPN2 (also known as TC-PTP) through deletion of the entire gene locus in ∼ 6% of T-cell acute lymphoblastic leukemia (T-ALL) cases. T-ALL is an aggressive disease of the thymocytes characterized by the stepwise accumulation of chromosomal abnormalities and gene mutations. In the present study, we confirmed the strong association of the PTPN2 deletion with TLX1 and NUP214-ABL1 expression. In addition, we found cooperation between PTPN2 deletion and activating JAK1 gene mutations. Activating mutations in JAK1 kinase occur in ∼ 10% of human T-ALL cases, and aberrant kinase activity has been shown to confer proliferation and survival advantages. Our results reveal that some JAK1 mutation–positive T-ALLs harbor deletions of the tyrosine phosphatase PTPN2, a known negative regulator of the JAK/STAT pathway. We provide evidence that down-regulation of Ptpn2 sensitizes lymphoid cells to JAK1-mediated transformation and reduces their sensitivity to JAK inhibition.

Description: The protein tyrosine phosphatase CD45, encoded by the PTPRC gene, is well known as a regulator of B- and T-cell receptor signaling. In addition, CD45 negatively regulates JAK family kinases downstream of cytokine receptors. Here, we report the presence of CD45 inactivating mutations in T-cell acute lymphoblastic leukemia. Loss-of-function mutations of CD45 were detected in combination with activating mutations in IL-7R, JAK1, or LCK, and down-regulation of CD45 expression caused increased signaling downstream of these oncoproteins. Furthermore, we demonstrate that down-regulation of CD45 expression sensitizes T cells to cytokine stimulation, as observed by increased JAK/STAT signaling, whereas overexpression of CD45 decreases cytokine-induced signaling. Taken together, our data identify a tumor suppressor role for CD45 in T-cell acute lymphoblastic leukemia.

Description: Abstract 42 Although BCR-ABL kinase inhibitors (TKIs) have revolutionized treatment of CML, they do not eradicate disease and treatment resistance can emerge. We recently identified Placental Growth Factor (PlGF) as an additional pathogenic factor in murine BCR-ABL1+ leukemia. PlGF induces proliferation of bone marrow stromal cells and of BCR-ABL1+ cell lines. PlGF is upregulated in blood (PB) and bone marrow (BM) of mice with BCR-ABL1+ leukemia and it's inhibition by a monoclonal antibody (αPlGF) significantly prolongs survival of mice bearing BCR-ABL1-unmutated and -T315I mutant blast crisis (BC) CML (Loges et al., Blood 2008; 112: abstract 1094). Now, we have further characterized the role of PlGF in disease progression, the mechanism of action of αPlGF and validated PlGF as novel target in human CML. We first quantified PB and BM PlGF protein levels at different time points after inoculation of BCR-ABL1 transduced BM cells and found significantly increasing PlGF levels upon disease progression. In line with those data, PB and BM PlGF protein levels correlated with numbers of GFP-marked leukemia cells (r=0.81; p=

Description: The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells. Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. Disclosures Levine: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Description: Cytokine-mediated signal transduction is critical to hematopoiesis, immune responses, and other physiological processes. Aberrant production and secretion of pro-inflammatory cytokines disturbs homeostasis and proper immune function and if persistent results in symptoms of chronic inflammation. Previous studies have illustrated the importance of JAK1 as an effector of cytokine signaling, including in immunological and neoplastic diseases such that selective JAK1 inhibition is currently being investigated in clinical trials. However, the role of Jak1 in hematopoietic stem cell (HSC) function has not been delineated. This has led us to investigate the impact of loss of Jak1 signaling on HSC function by developing a novel conditional Jak1 knockout allele (Fig. 1a). Mice with conditional deletion of Jak1 in the hematopoietic system (hereafter referred to as Jak1 KO) are characterized by leukocytosis (Jak1 KO avg. 6.34K/ul, Jak1 WT avg. 10.76K/ul, P