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Synthetic lethality occurs when a single gene alteration is compatible with cell viability, but an additional co-occurring genetic alteration leads to cell death. In the context of cancer therapy, synthetic lethality can occur through the inhibition of a target that is selectively essential to tumors harboring a specific genetic alteration. Gene paralog pairs represent one promising class of synthetic lethal cancer targets, wherein the function of one paralog is lost in tumor cells, rendering them dependent on the remaining paralog to carry out an essential cellular process. To identify essential gene paralog pairs as starting points for drug discovery programs, we mined publicly available CRISPR genetic loss-of-function data and associated molecular datasets collected across a diverse panel of cancer cell lines. We first identified pairs of gene paralogs where one paralog was essential in a subset of cell lines, and then filtered these genes based on function, known literature, enrichment in specific lineages and integration of external datasets. These efforts identified VPS4A as a synthetic lethal target in cancers harboring copy number loss of VPS4B. VPS4A and VPS4B are highly homologous AAA ATPases that carry out multiple essential cellular processes including nuclear membrane remodeling and endosomal membrane biogenesis. VPS4B loss occurs as a passenger deletion during loss of the tumor suppressors SMAD2 and SMAD4. Loss of VPS4B creates a genetic dependency on VPS4A to drive essential VPS4-dependent processes. VPS4B deletion occurs at a frequency of up to 3% in multiple solid tumor types including esophageal, head and neck, pancreatic and colorectal cancers. To further explore the potential of VPS4A as a therapeutic target in VPS4B-deleted tumors, we first validated the synthetic lethal relationship between VPS4A/B using isogenic cell line pairs. HCT116 cells with an engineered homozygous loss of VPS4B, but not wild-type HCT116 cells, showed profound cell kill in response to genetic silencing of VPS4A. Moreover, simultaneous siRNA-mediated knockdown of VPS4A and VPS4B resulted in cell death across a panel of cancer cell lines (e.g. H1975, Panc0403), while knockdown of either gene alone was compatible with cell viability. Encouraged by these results, we profiled several previously reported small-molecule inhibitors of VPS4A (e.g. DBeQ and MSC1094308) in a suite of biochemical assays. Notably, these molecules were inactive against VPS4A. We have discovered a novel series of VPS4A inhibitors and are advancing this inhibitor series through lead optimization. Potent, selective, and pharmacologically active VPS4A inhibitors are expected to be well tolerated and have strong single-agent activity in tumors bearing VPS4B homozygous deletions. Citation Format: Meredith Kuo, Jason Chen, Sacha Holland, Eugene Lurie, An-Angela Ngoc Van, Francesco Parlati, Tayna Santos, Eric Sjogren, Natalija Sotirovska, Susanne Steggerda, Andrew MacKinnon. Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers [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 1816.

Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor–stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. Cancer Res; 70(4); 1544–54

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA–mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.

Abstract 940 Novel targets with potential to improve treatment of AML are urgently needed. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and circumstantial evidence in literature links Axl expression to AML pathobiology. However, a systematic study of the role of TAMRs and of their common ligand growth arrest-specific oncogene 6 (Gas6) in AML is missing until now. Therefore, we set out to first study the prognostic impact of Gas6 and TAMRs in a uniform cohort of cytogenetically normal AML patients (n=112) entered into the multicenter treatment trial AML SHG0199. By quantitative RT-PCR we determined pre-treatment expression levels of Gas6 and TAMR and subsequently correlated gene expression data with clinical data. We found expression of Axl, Tyro3, Mer and Gas6 in 57%, 79%, 85% and 90% of patients, respectively. Upon correlation of expression data with survival data of patients who expressed the respective gene, only Axl expression level represented an independent prognostic factor after correcting for age, sex, ECOG performance status, bone marrow blast count and white blood cell count at presentation. Patients expressing Axl above the median experienced a significantly shorter OS than patients expressing Axl below the median (28 months vs. not reached; p=0.037; median follow up 5.45 years). Axl expression levels were independent of patient age, sex and FAB type. Based on prognostic significance of Axl expression, we focused on evaluating the potential of Axl as therapeutic target in AML. First we analyzed a panel of different AML cell lines including HL-60, MV4-11, OCI-AML-5, KG-1, THP-1, U937 and Kasumi-1 for target expression. All cell lines except for Kasumi-1 showed expression of Axl and Gas6 was expressed in MV4-11, OCI-AML-5 and U937. For further studies we focused on MV4-11 and OCI-AML-5 cells. To investigate effects of Axl inhibition on leukemia pathobiology, we utilized R428, a well-characterized small molecule inhibitor of Axl. Application of R428 monotherapy inhibited proliferation of MV4-11 and OCI-AML-5 cells in a dose-dependent manner with an IC50 of 1.74 uM and 1.35 uM, respectively. Furthermore, R428 dose-dependently induced apoptosis in both cell lines. Because of the worse outcome of AML patients expressing high levels of Axl, we hypothesized that Axl might be implicated in resistance to chemotherapy. Indeed, treatment of MV4-11 and OCI-AML-5 cells with doxorubicin or cytarabine upregulated their Axl expression. Based on these data we conducted matrix experiments of R428 in combination with both drugs. Consistent with our hypothesis, R428 significantly enhanced chemosensitivity of MV4-11 and OCI-AML-5 cells. To investigate whether Gas6 is necessary for the pro-leukemic actions of Axl, we applied soluble Axl, which neutralizes Gas6, to MV4-11 and OCI-AML-5 cells, alone and in combination with chemotherapy and/or R428. In absence of Gas6 both the effect of R428 monotherapy and the additive effect of concomitant chemotherapy and Axl-inhibition were abrogated, hence the therapeutic effect of R428 specifically depends on disrupting Gas6-Axl interaction. Next, we analyzed different Axl-downstream signaling pathways in order to elucidate via which pathway R428 acts in MV4-11 and OCI-AML-5 cells. We could show that R428 dose-dependently inhibits Axl phosphorylation. Furthermore, R428 inhibits phosphorylation of Akt, which is increased in response to chemotherapy. Thus, the chemosensitizing effect of R428 is at least partly mediated by inhibiting activation of Akt. Encouraged by those data we investigated therapeutic potential of R428 in vivo by using the MV4-11 xenograft model. Mice bearing established tumors with a mean size of 294 mm3 (n=17) were randomized and subsequently treated with 75 mg/kg R428 or vehicle control twice daily by oral gavage. Treatment with R428 completely inhibited MV4-11 growth when compared to control treatment (end stage tumor size 1185 mm3 + 253 mm3vs. 184 mm3 + 46 mm3, respectively; n=8/9; p=0.0009). In summary, our data indicate that Axl represents a novel prognostic marker in CN AML. Therapeutic targeting of Axl by R428 exerts anti-leukemic activity in vitro and in vivo, thus treatment with R428 might open up novel therapeutic avenues in AML, potentially useful alone or in combination with chemotherapy. Disclosures: Fiedler: Pfizer: Research Funding.

Background: Metastasis and drug resistance are the major causes of mortality in patients with pancreatic ductal adenocarcinoma (PDA). Once developed, the progression of PDA metastasis is virtually unstoppable with current therapies. Accumulating evidence suggests important roles for the receptor tyrosine kinase AXL in cancer progression, invasion, metastasis, drug resistance, and patient mortality. Given the critical influence of metastasis on the clinical history of PDA, we hypothesize that pharmacological inhibition of AXL might inhibit tumor growth and prevent PDA metastasis. Methods: We investigated the AXL expression in a panel of human pancreatic cancer xenografts using Affymetrix gene array as well as qRT-PCR. We conducted a comprehensive preclinical trial to determine whether AXL targeted monotherapy, and in combination with gemcitabine, was effective in blocking PDA tumor progression and metastasis using subcutaneous as well as orthotopic pancreatic cancer models. Results: R428, a potent selective small molecule inhibitor of AXL, inhibited proliferation of pancreatic cancer cell lines in a concentration-dependent manner. A combination of R428 with gemcitabine could significantly inhibit primary tumor growth compared to gemcitabine treated mice. Notably, R428 alone and in combination with gemcitabine was remarkably effective in preventing the development of metastasis to organs and lymph nodes as compared to gemcitabine treatment in a highly aggressive orthotopic model. Conclusions: The data presented here provide strong evidence for the therapeutic development of AXL inhibitors as anti-metastatic agents in pancreatic cancer. A combination treatment of R428 and gemcitabine was highly effective in controlling the primary tumor growth and preventing metastatic spread. The novel therapeutic approach tested here has potential therapeutic implications for improving the current standard of care for PDA patients and to contribute towards making PDA a more manageable disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2011-2546

Abstract 54 B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B-lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. Deregulated expression of protein kinases by gene deletion, mutation, or amplification has been found to be important for tumor initiation and progression involving cancer cell proliferation, survival, motility, and invasiveness as well as tumor angiogenesis and chemotherapy resistance. Because of their critical functions in oncogenesis, protein kinases have been at the forefront of targeted cancer therapy development since the 1980s. A novel receptor tyrosine kinase (RTK) Axl has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Indeed, our recent investigation (ASH 2009, Abstract #2368) identified Axl to be constitutively phosphorylated in CLL B-cells obtained from a majority of CLL patients. We wished to examine whether the constitutively phosphorylated Axl plays a pivotal role in regulating leukemic CLL B-cell survival and could be a potential target to treat this disease. We used freshly isolated purified CLL B-cells after obtaining informed written consent from the patients. Expression status of various kinases in relation to Axl expression and their functional involvement with this novel RTK to drive downstream cell survival signaling pathway(s) were analyzed in CLL B-cell lysates by immunoprecipitation/Western blot analyses using specific antibodies. Finally, we evaluated the functional importance of Axl in CLL by employing two RTK inhibitory agents (Bosutinib [SKI-606] and R428) of different specificity and affinity for receptor tyrosine kinases in order to target Axl and determined the impact on apoptotic/cytotoxic effects on CLL B-cells by flow cytometric analysis. In some experiments, we used bone marrow stromal cells to evaluate if they could diminish the impact of the RTK agent R428. To further our initial examination of Axl status in CLL, we assessed the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B-cells by Western blot analysis. Indeed, we detected differential levels of P-Axl in CLL B-cells (n=10). Further analysis revealed that the expression of P-Axl was associated with co-expression of other constitutively phosphorylated kinases/enzymes including Src family kinases (SFKs), PI3K, SyK/ZAP70 and PLC-g2 in CLL B-cells. Importantly, we found by immunoprecipitation of Axl and subsequent Western blotting that these intracellular signaling molecules were complexed with P-Axl in primary CLL B-cells. Further analysis using a siRNA approach targeting Axl suggests that phosphorylation of Axl is an upstream event of SFK-activation in CLL B-cells indicating a pivotal role of Axl in regulating CLL B-cell survival. Finally, when Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, SKI-606 (dose range 2.5–20mM) or the specific-inhibitor of Axl, R428 (dose range 0.5–10 mM) robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner (Figure). Although both RTK inhibitory agents showed clear induction of apoptosis in CLL B-cells, R428 was found to be the most potent in inducing apoptosis exhibiting an IC50 ∼4-fold lower than SKI-606. Furthermore, we have observed that R428 could target P-Axl resulting in inhibition of SFK-activation further suggesting that Axl regulates SFK-activity in CLL B-cells. Interestingly, R428-induced apoptosis in CLL B-cells (doses of 1.0 mM and 2.5 mM) was significantly inhibited when these leukemic cells were co-cultured with bone marrow stromal cells, but when we tested a higher dose (5 mM) of R428 this stroma-mediated protection was overcome. Further studies are underway to test R428 in combination other chemotherapeutic agents. Thus, we have identified a novel RTK, Axl, in CLL B-cells which appears to work as a docking site for multiple non-RTKs and drives cell survival signals. These findings indicate a unique therapeutic target for CLL treatment. Importantly, there are currently several Axl inhibitory agents that are available to be tested in CLL patients. Disclosures: Holland: Rigel: Employment.

Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

Limited options provided by the current standard of care for the patients suffering from myeloproliferative diseases (MPDs) prompted an extensive search for the underlying molecular mechanisms of these disorders. Recent discovery of a single activating mutation (V617F) in JAK2 kinase gene associated with the development of the polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) opened up a possibility to develop highly targeted therapies against these debilitating ailments. To that end, we engineered cytokine-independent Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F -dependent proliferation. We confirmed the ability of hit compounds to inhibit proliferation of JAK2-dependent tumor cell lines using UKE-1 and SET-2 cells carrying the V617F JAK2 mutation. A FACS-based phosphoSTAT5 assay was then used to demonstrate that the hits directly targeted mutant JAK2. JAK3 activity of each compound was evaluated in IL-2-dependent CTLL-2 cell line using phosphoSTAT5 FACS and proliferation assays. To avoid hits with nonspecific antiproliferative activity, the hits were tested in JAK2-independent MOLT4, A549 and H1299 cell lines. Compound hits with the desirable properties were further evaluated for their ability to inhibit JAK2, JAK3 and other kinases in the context of T cell, B cell, or mast cell activation using a variety of cell-based assays as well as in the in vitro biochemical assays. We identified a number of compounds that potently inhibit growth of the two V617F mutant cell lines with EC50s varying from 20 to 500 nM, but do not affect proliferation of control cell lines MOLT4, A549 and H1299 to the same degree. These compounds induce strong and highly specific suppression of STAT5 phosphorylation with IC50s of 10 to 200 nM in SET-2 and V617F JAK2 expressing Ba/F3 cells. One of the hits with the desirable biological and pharmacokinetic profiles was further evaluated in V617F JAK2 Ba/F3 engraftment mouse model where it demonstrated significant extension of survival at 150 and 200 mg/kg bid. Such potent JAK2 inhibitors could become the basis for the next generation of compounds targeting JAK2-dependent myeloproliferative diseases.