JAK Inhibitor Ruxolitinib Research Articles

Abstract 4699: Superior lethal activity of single agent BET protein PROTAC compared to bromodomain inhibitor or in combination with JAK inhibitor against post-myelofibrosis secondary AML cells

Abstract Myeloproliferative neoplasm, myelofibrosis (MPN-MF), exhibits increased JAK-STAT signaling and often progresses (∼15-20%) to AML (sAML). JAK inhibitor (JAK-I) ruxolitinib (Rux) or standard induction chemotherapy is only modestly active against sAML, culminating in treatment-refractory relapse. Genetic alterations commonly documented in sAML include the co-occurrence of JAK2 V617F and mutant TP53. Here, we demonstrate that treatment with ARV-825 (Arvinas, Inc.), a BET (bromodomain and extraterminal) protein PROTAC (proteasome activating chimera) which degrades (through the proteasome) BET proteins by binding and recruiting E3 Ubiquitin ligase cereblon activity to them, caused efficient, and prolonged depletion of the levels of the BET protein BRD4. In contrast, treatment with the bromodomain inhibitors (BET-Is) JQ1 or OTX015 induced the levels of BRD4. ARV-825 treatment also mediated greater and more sustained attenuation than OTX-015 of the mRNA and protein expressions of BCL-xL, CDK4/6, PIM1, p-STAT5 and p-STAT3 levels, while concomitantly inducing p21 and p27 in the cultured sAML (HEL92.1.7 and SET2) cells. This correlated with high level of apoptosis in the cultured and patient-derived post-MPN-MF sAML cells, with relative sparing of the normal CD34+ progenitor cells. Compared to treatment with each agent alone, co-treatment with ARV-825 and the JAK-Is Rux (100 to 1000 nM) or pacritinib (250 to 1000 nM), was synergistically more lethal against the cultured sAML cells (CI of < 1.0 on the isobologram analyses). Co-treatment with ARV-825 and Rux caused a marked inhibition of p-STAT5, p-STAT3, MYC, CDK4/6, BCL-xL and PIM1 in the sAML cells. HEL92.1.7 and SET2 cells harbor JAK2 V617F as well as express mutant TP53 (M133K in HEL92.1.7 and R248W in SET2 cells). Co-treatment with ARV-825 or JQ1 and the heat shock protein (HSP) 90 inhibitor AUY922, which is known to down-regulate the levels of mutant-TP53, JAK2, c-RAF, p-STAT5, p-STAT3 and p-AKT, was synergistically lethal against HEL92.1.7 and SET2 cells. We have isolated JAK-I-resistant HEL92.1.7 cells (> 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK-I. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to both ARV-825 and AUY922. Furthermore, co-treatment with ARV-825 and AUY922 was also synergistically lethal against HEL/JIR cells (CI < 1.0). Taken together, these findings demonstrate that ARV-825 is a highly active agent alone or in combination with JAK-Is against post-MF sAML cells expressing mutant p53. Combined therapy with ARV-825 and an HSP90 inhibitor is synergistically lethal against sAML cells resistant to JAK-Is. Based on these finding, in vivo testing of the BRD4 PROTAC-based combinations against post-MF sAML cells is warranted. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Jing Lu, Yimin Qian, Kanak Raina, Baohua Sun, Stephanie S. Krieger, Simrit Parmar, Srdan Verstovsek, Kapil N. Bhalla. Superior lethal activity of single agent BET protein PROTAC compared to bromodomain inhibitor or in combination with JAK inhibitor against post-myelofibrosis secondary AML cells. [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 4699.

546 Gene expression signatures and ALADIN score correlates with response of alopecia areata patients to treatment with JAK inhibitors

Alopecia areata (AA) is an autoimmune disease in which the hair follicle is the target of immune attack and manifests clinically as hair loss. The more extreme forms of the disease can result in lifelong alopecia with complete loss of scalp, face and body hair. Currently, there are no FDA-approved drugs for AA, and response to treatments are highly variable and often disappointing. Biomarkers for the purposes of precise quantitative tracking of disease severity, disease course prediction, and response to drug are valuable tools in the evaluation of novel treatments in clinical trials for AA. Previous studies by our group, including large genome-wide association studies and meta-analyses, gene expression analyses conducted on human AA scalp skin biopsies and animal models, and targeted cytokine antibody treatments in the murine form of AA, provided a strong rationale for targeting Janus kinase signaling intermediates for the purposes of treatment. Our center recently completed two pilot, open-label clinical trials of the JAK inhibitors ruxolitinib and tofacitinib, for the treatment of AA. Using previously collected whole genome gene expression data collected from a large cohort of patients with AA as well as normal controls, a multifactorial metric we termed the Alopecia Areata Disease Activity Index, or ALADIN, was created. In this study, we used the ALADIN metric to measure responses of AA patients enrolled in our clinical trials of JAK inhibitors. This tool tracked with clinical score and demonstrated strong correlation with response to treatment. Furthermore, ALADIN component genes, which include GZMA, PRF1, CXCL9, CXCL10 as well as hair-keratin associated genes, differentiated eventual responders from nonresponders at baseline in some cases. ALADIN is a powerful biomarker tool that will be useful in clinical trials of novel treatments for AA, particularly in larger randomized clinical trials.

Abstract B31: EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma

Abstract Pharmacological inhibition of the histone lysine methyltransferase EZH2 has emerged as a therapeutic strategy for the treatment of non-Hodgkin Lymphoma (NHL), in particular for cases with mono-allelic mutations in the EZH2 catalytic domain. Potent, selective EZH2 small molecule inhibitors have achieved tumor regression in mutant EZH2-containing preclinical lymphoma models and several of these inhibitors are currently engaged in cancer-focused clinical trials. Here, we show that the presence of EZH2 mutations do not always confer EZH2 inhibitor sensitivity. We discovered that EZH2 is usurped by lymphoma cells to attenuate JAK/STAT signaling and to repress pro-apoptotic interferon response genes. EZH2 inhibition results in the broad induction of interferon-stimulated genes (ISGs) in several phenotypically sensitive NHL cell models. In mutant EZH2-containing insensitive models, EZH2 inhibitors synergize specifically with type I interferons (IFNs) in vitro and in vivo to induce ISGs and apoptosis. The profound combinatorial activity of EZH2 inhibitors and type I IFNs is not restricted to NHL models with mutant EZH2 and, is preferentially observed in models that are not affected by either single agent. Molecular consequences of EZH2 inhibitor and type I IFN combinatorial treatment include STAT1 activation, binding of STAT transcription factor complexes to ISGs, reduction of H3K27me3 levels and increase of H3K4me3 levels at ISG transcriptional start sites, and ISG transcriptional activation. The molecular and anti-proliferative effects of the combination can be suppressed by inactivation of the JAK/STAT signaling pathway, by using the JAK inhibitor ruxolitinib. We suggest that EZH2 inhibitors can be therapeutically combined with type I IFNs for the treatment of NHL. Citation Format: William D. Bradley, Chih-Chi Yuan, Feng Zhao, Christina Lee, Kaylyn E. Williamson, Shilpi Arora, Charlie Hatton, Barbara M. Bryant, Patrick Trojer. EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B31.

Pacritinib, a Dual FLT3/JAK2 Inhibitor, Reduces Irak-1 Signaling in Acute Myeloid Leukemia

Background: A momentous challenge in the treatment of acute myeloid leukemia (AML) is the significant molecular heterogeneity in driving abnormalities and rapid emergence of resistance. Successful clinical translation of novel targeted agents has been impeded by an incomplete understanding of the genetic drivers and the role of the bone marrow microenvironment that modulates response to therapy. There is thus a need for further investigation of the signaling mechanisms contributing to disease pathogenesis. We previously identified growth dependency on interleukin-1 (IL-1) signaling in >50% of the 90 primary AML samples tested. IL-1 signals through IL-1 receptor-associated kinase (IRAK1), a major mediator of innate immunity and inflammatory responses and a potentially critical therapeutic target in hematopoietic malignancies. These results suggest that targeting the commonly dysregulated IL-1 signaling pathway would be therapeutically beneficial to AML patients. However, there is no clinically viable molecule available to selectively inhibit IL-1 signaling. Here we uncovered that pacritinib, a dual FLT3/JAK2 inhibitor, has high specificity and sensitivity to target IRAK1 in various hematopoietic malignancies, including AML. Methods: We used a combination of biochemical, structural biology, and functional approaches to elucidate the mechanism of action of pacritinib and establish its sensitivity and specificity for target inhibition. Kinome screening analysis against 429 recombinant kinases in the presence of 100 nM pacritinib (approximately 50% of the steady state concentration of free pacritinib at the phase 3 dose of 400 mg QD) was followed by titration (1-100 nM) against those kinases that were >50% inhibited at 100 nM. Because pacritinib is an established dual FLT3/JAK2 inhibitor, we then compared the efficacy of pacritinib to the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 16 AML cell lines and 25 primary AML samples harboring various genetic lesions. The effects of drug treatments were evaluated on cell viability, survival and downstream signaling. Patient samples having IC50below 1000 nM were considered responsive. To validate the selectivity of pacritinib for binding IRAK1, we identified key interacting residues by molecular docking simulation and performed targeted mutagenesis studies. Results: Pacritinib inhibited the activity of FLT3 and all JAK family members except JAK1 at IC50 values of Conclusions: Pacritinib is a specific inhibitor of IRAK1 that blocks IL-1 signaling in AML-derived cell lines and primary patient samples. To our knowledge, pacritinib is the first clinically viable molecule to robustly and selectively inhibit IRAK1. These findings may be extended to a variety of cancer types with a dependence on IRAK1 kinase. Disclosures Druker:Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; ARIAD: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees; AstraZeneca: Consultancy; McGraw Hill: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; Leukemia & Lymphoma Society: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Singer:CTI BioPharma, Corp: Employment, Equity Ownership. Agarwal:CTI BioPharma: Research Funding.

Lysine-Specific Histone Demethylase, LSD1, (KDM1A) As a Novel Therapeutic Target in Myeloproliferative Neoplasms

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. [Display omitted] DisclosuresRiehnhoff:Imago: Employment, Equity Ownership. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

The JAK Inhibitor Ruxolitinib Is Effective in Treating T Cell Acute Lymphoblastic Leukemia with Gain of Function Mutations in IL-7R Alpha

Acute lymphoblastic leukemia (ALL) results from transformation of immature B or T cells, and is the most common pediatric cancer. Though the current cure rate of ALL is 80-90%, it is important to understand the underlying biology of ALL in order to develop refined therapies for patients who fail to respond to conventional chemotherapy as well as to reduce its toxicity. The IL-7 receptor (IL-7R) signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R alpha. These mutations promote homodimerization of IL-7R alpha subunits, resulting in constitutive activation of this pathway via Janus Kinase 1 (JAK1). As the JAK-STAT pathway is downstream of the IL-7 receptor, we hypothesized that JAK inhibitors could be used to treat T-ALL patients with IL-7R mutations. To demonstrate this, we first established a cell line model of T-ALL driven by constitutive IL-7R signaling. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7R alpha derived from a patient sequence. These cells termed "D1_hIL7R_P1" are also GFP+, which allows us to monitor the proliferation of the cells in vivo.I have showed that D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Ruxolitinib, a JAK1 inhibitor, inhibits survival, proliferation, and STAT5 activation of D1_hIL7R_P1 cells in vitro. To treat this leukemia in vivo, I administered ruxolitinib for 5 days at a dose of 150 mg/kg to mice starting 8 days after D1_hIL7R_P1 engraftment. Tissues were then harvested for analysis of GFP+ cells as a measure of leukemic burden via flow cytometry. Ruxolitinib reduced leukemic cells from 15% in the blood (as demonstrated in untreated mice) to 5%, from 30% to 10% in the spleen, and 40% to 20% in the lungs. With these promising results, I have acquired T-ALL patient samples that can be xenografted into NSG mice. TALL#5 cell line successfully engrafts in 30 days and can be detected in the bone marrow, spleen, and peripheral blood by flow cytometry. Additionally, TALL#5 expresses human IL-7R alpha and is sensitive to ruxolitinib treatment in vitro. I have also identified two T-ALL cell lines, DND41 and KOPTK1, which express high levels of IL-7R alpha. These cell lines are also potential candidates for studying the effects of ruxolitinib on T-ALL in NSG mice. TALL#5, DND41, and KOPTK1 will be engrafted into mice, and after leukemia is established, mice will be treated with ruxolitinib. If successful, this will give us insight into the effectiveness of JAK inhibitors in treating ALL, and which patients can be recruited for future clinical trials. DisclosuresOff Label Use: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved for myelofibrosis. My experiments involve pre-clinical studies looking at the efficacy of ruxolitinib in leukemic mice..

Subgroups of T-Cell Prolymphocytic Leukemia (T-PLL) Discovered By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients.Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping.To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases.Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort.Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status.Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. [Display omitted] DisclosuresKoschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Superior Activity of BET Protein Bromodomain Antagonist-Based Combination with JAK Kinase, PIM Kinase or Heat Shock Protein 90 Inhibitor Against Post-MPN-MF sAML Cells

Myeloproliferative Neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of the JAK-STAT signaling and often progress (~20%) to AML (sAML). As a single agent, JAK1&2 inhibitor ruxolitinib confers significant clinical benefit in MPN-MF, but exhibits modest activity in sAML, which is also incurable with standard anthracycline and Ara-C-based chemotherapy. This supports the rationale to develop and test novel combination therapies for post-MPN-MF sAML. Genetic alterations documented in sAML include those in JAK2, MPL or calreticulin (CALR) gene, as well as in TP53, TET2, ASXL1, IDH1&2, SRSF2, RUNX1, MYC, PTPN11, NRAS, and SETBP1 genes. Variant allelic frequency analyses demonstrated a common co-occurrence of JAK2V617F and mutant TP53 in the dominant clones of sAML. We and others have previously reported that treatment with BET (bromodomain and extra-terminal) protein bromodomain antagonists (BA) results in growth arrest, differentiation and apoptosis of AML cells (Mol Cancer Ther 2014,13:2315). In the present studies, we demonstrate that treatment with JQ1 but not its inactive enantiomer (R-JQ1) dose-dependently (100 to 2000 nM) mediates growth inhibition and apoptosis of the cultured (HEL92.1.7, SET2 and UKE1 cells) and primary (p) post-MPN-MF sAML cells. JQ1 treatment reduced the promoter occupancy of BRD4 and RNA polymerase II on MYC, BCL2 and IL7R promoters, attenuated the mRNA and protein expressions of BCL2, BCL-xL, MYC, CDK4/6 and PIM1, and repressed the pSTAT5 and pSTAT3 levels, while concomitantly inducing the levels of HEXIM1, p21, p27 and BIM in the sAML cells. Following engraftment of NOD/scid/IL-2Rγ null) (NSG) mice with HEL92.1.7 cell xenografts, treatment with JQ1 (50 mg/kg/day, administered IP daily x 5 days per week x 3 weeks) also significantly improved the median survival of the mice (p < 0.01). Compared to treatment with each agent alone, co-treatment with JQ1 and ruxolitinib (100 to 1000 nM) or pacritinib (250 to 1000 nM), which is also a clinically active JAK2, JAK2-V617F and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1, was synergistically lethal against the cultured sAML cells (Combination indices of < 1.0 on the isobologram analyses). Additionally, co-treatment with JQ1 and ruxolitinib caused a marked inhibition of pJAK2, pSTAT5, pSTAT3, MYC, CDK4/6, BCL-xL and PIM1 in the sAML cells. Co-treatment with JQ1 and the pan-PIM kinase inhibitor AZD1208 (500-3000 nM), which inhibits the PIM kinase substrates BAD, p70S6 kinase and 4EBP1, also synergistically induced apoptosis of the cultured sAML cells (CI < 1.0). HEL92.1.7 and SET2 cells are not only positive for the JAK2V617F mutation, they also express mutant TP53 (M133K in HEL92.1.7 and R248W in SET2 cells). Co-treatment with JQ1 and the heat shock protein (HSP) 90 inhibitor AUY922 (2.5-20 nM), which is known to down-regulate the levels of mutant-TP53, JAK2, c-RAF, pSTAT5, pSTAT3 and pAKT, is synergistically lethal against HEL92.1.7 and SET2 cells. We have isolated JAK inhibitor-resistant HEL92.1.7 cells (> 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK inhibitor (Clin Cancer Res 2011;17:7347). Compared to the parental HEL92.1.7, HEL/JIR cells are highly and collaterally sensitive to AUY922. HEL/JIR cells also remain sensitive to JQ1-induced apoptosis. Importantly, co-treatment with JQ1 and AUY922 is also synergistically lethal against HEL/JIR cells (CI < 1.0). Taken together, these findings highlight the pre-clinical activity of BA-based combination with JAK inhibitors (ruxolitinib or pacritinib), PIM kinase inhibitor (AZD1208) or HSP90 inhibitor (AUY922) against JAK kinase inhibitor-sensitive sAML, as well as of the BA-based combination with HSP90 inhibitor or pan-PIM kinase inhibitor against JAK inhibitor-resistant sAML cells. Collectively, these findings also support further in vivo testing of these BA-based combinations against sAML cells. DisclosuresNo relevant conflicts of interest to declare.

Stimulation of the B-Cell Receptor (BCR) Induces Successive Activation of STAT3 and Nuclear Factor-Kappa-B in CLL Cells

Abstract Introduction: While in CLL cells phosphorylation of STAT3 on serine 727 residues is constitutive, phosphorylation of STAT3 on tyrosine 705 residues is inducible. Cytokines, such as IL-6, or IgM antibodies that activate CLL cells' BCR, induce tyrosine phosphorylated (p) STAT3. However, whereas IL-6 induces tyrosine pSTAT3 phosphorylation within 15 minutes, IgM induces pSTAT3 within ≥ 2-4 hours. The reason for the delayed IgM-induced phosphorylation is unknown. Like STAT3, the transcription factor NF-κB is constitutively activated in CLL cells and stimulation of the BCR activates NF-κB. Whether BCR stimulation upsurges NF-κB's transcriptional activity has not been elucidated. Because IL-6 is an NF-κB-target gene and, like IL-6, IgM antibodies induce tyrosine pSTAT3, we wondered whether prolonged stimulation with IgM antibodies induces tyrosine pSTAT3 via NF-κB-mediated induction of IL-6 in CLL cells. Methods: We incubated peripheral blood CLL cells in the presence or absence of IgM antibodies or IL-6, and harvested the cells at different time points. Total RNA was extracted using TRIzol (Life technology), cDNA was synthesized with Super Script First synthesis System for RT-PCR (Invitrogen), and NF-κB-target gene expression was quantified using RT-PCR (Invitrogen Life Sciences). To measure the levels of tyrosine pSTAT3 we used flow cytometry and to assess binding of NF-κB (p65) to DNA we utilized an electromobility shift assay (EMSA) using an NF-κB-binding site labelled DNA probe. Results: The transcriptional activity of NF-κB was studied using a PCR array that profiles the expression of 83 NF-κB-target genes. To reduce the 'noise' from stochastic variability in gene expression we first identified a core of genes that are expressed in cells from all patients' samples. To that aim we ranked the Ct values in each array and considered all genes that were amplified earlier than the cycle in the 75th percentile. Using this approach we identified 35 genes (42% of genes represented in the array) that were amplified in all 6 patients' samples. Annotation analysis revealed that the key pathways common to these 35 genes included 'Positive regulation of the NF-κB cascade', 'Inflammation' and 'Negative regulation of apoptosis'. Applying stringent criteria we identified 5 genes common to all cases that were amplified prior to the cycle representing the 25th percentile. Most amplified genes detected in all samples prior to stimulation (28/35, 80%) were also detected after 4 h of IgM stimulation, confirming that NF-κB is constitutively activated in CLL cells. However, 19 addition genes (19/83, 23%of the genes in the array) were detected in all IgM-stimulated but not in unstimulated cells. Remarkably, IL-6 was detected in all cases only after IgM stimulation. Furthermore, the delta-delta Ct method identified an IgM-induced time-dependent increment in IL-6 and IL-8, suggesting that IL-6 expression is dependent on stimulation of the BCR. Indeed IL-6 neutralizing antibodies significantly reduced the levels of tyrosine pSTAT3 in CLL cells incubated for 18 h with IgM antibodies. In addition, EMSA studies using CLL cells from 4 different patients showed that stimulation of the BCR with IgM antibodies increased the binding of NF-κB to DNA in a time-dependent manner. Moreover, the JAK2 inhibitor Ruxolitinib attenuated the NF-κB-DNA binding, suggesting that long exposure to IgM antibodies induces activation of NF-κB, a process mediated in part by IL-6 that activates the JAK2/STAT3 pathway. Conclusions: The BCR of CLL cells is stimulated in the bone marrow and lymph nodes. However, whereas the immediate effects of BCR stimulation have been excessively studied, the successive effect BCR stimulation is poorly understood. We found that stimulation of the BCR induces tyrosine phosphorylation of STAT3 via NF-κB-mediated induction of IL-6, a process that requires protracted BCR stimulation. Although NF-κB is constitutively activated in CLL cells, continuous activation of the BCR further activates NF-κB. Continuous stimulation of the BCR increases the levels of IL-6 that, upon binding to its receptor, activates STAT3 that in turn activates NF-κB. Taken together, our data suggest that agents, such as Ruxolitinib, that inhibit the successive effects of BCR activation, would become effective therapeutic agents in CLL. Disclosures Rozovski: Novartis: Other: Advisory board. Wierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy.

Increased Alternative Macrophage-2 (M2) Polarization with Trib1 Gene over Expression in Myeloma Patients with Progressive Disease and Jak2 Inhibitors Reduce M2 Polarization

Introduction: The bone marrow (BM) microenvironment plays an important role in multiple myeloma (MM). The BM niche is composed of multiple cell types including macrophages. Macrophages polarize into pro-inflammatory macrophage-1 (M1) or alternative M2 states that promote tumor growth and metastasis. We evaluated the proportion of M2 macrophages in BM from MM pts either showing complete response (CR) or progressive disease (PD), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in macrophages, we also evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM.Methods: Using immunofluorescence (IFC), we determined the proportion of M1 and M2 macrophages in BM biopsies and aspirates from MM pts with PD or CR. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells. MM BM aspirates were also examined using flow cytometric analysis (FCA). Human monocytes isolated from healthy subjects or the THP1 monocyte cell line were co-cultured with MM cell lines (RPMI8226 and U266) or primary MM tumor cells. The effects of RUX at low concentrations (IC20) on M2 polarization were determined. The percentages of M1 and M2 macrophages were determined using FCA. Total RNA was extracted from monocytes. Quantitative PCR was measured with TaqMan technology. For the in vivo studies, human MM tumors (LAGκ-2) were surgically implanted into the left superficial gluteal muscle of SCID mice and tumor volume measured on a weekly basis.Results: The proportion of M2 macrophages (CD36+/ARG1+) was markedly increased in BM biopsies or mononuclear cells from MM pts with PD compared with those in CR using IFC staining. FCA also showed the percentage of M2 macrophages in BM was significantly increased in MM pts with PD (n=25) compared to those in CR (n=10; P=0.005) whereas there was no difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM pts with PD compared to those in CR. Trib1 gene mRNA levels were higher among pts with PD compared to those in CR whereas the gene expression of Trib2 and Trib3 was not different. Next, we co-cultured MM cell lines (U266) or fresh MM BMMCs with purified healthy human monocytes for one week. The percentage of M2 cells markedly increased and the proportion of M1 cells decreased. Trib1 gene expression increased during co-culture whereas there was no change in expression of the other two Tribs. When direct cell-to-cell contact occurred between the MM tumor cells and the monocytes, the percentage of M2 macrophages markedly increased. We investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. After exposure to a low concentration of RUX, the percentage of M2 cells decreased when the monocytes were co-cultured with MM tumor cells. Trib1 gene expression of the monocytes treated with RUX was also notably reduced compared with cells not treated with the JAK2 inhibitor. Using our human MM xenograft model LAGκ-2, RUX (1.5mg/kg) reduced tumor growth and decreased the proportion of M2 macrophages in the tumor tissue of MM tumor-bearing SCID mice.Conclusion: M2 cells are present at high levels in BM derived from MM pts with PD compared to those in CR, MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression. This induces monocyte differentiation into M2 macrophages that support MM tumor cell growth.. Notably, the JAK2 inhibitor RUX inhibits both M2 macrophage polarization and Trib1 gene expression in MM, and reduces tumor growth in SCID mice bearing human MM. These results suggest that RUX may be effective for treating MM pts. DisclosuresNo relevant conflicts of interest to declare.

The JAK Inhibitor Ruxolitinib Impairs Dendritic Cell Migration Via Off-Target Inhibition of Rock

Introduction: Ruxolitinib is a JAK inhibitor that was recently approved for treatment of primary and secondary MF and shows impressive symptom control by suppression of inflammation. The observed effects are irrespective of the JAK mutational status. Ruxolitinib is also a promising drug for treatment of acute and chronic GvHD (Spoerl et al., Blood 2014 and Zeiser et al., Leukemia, in press). We recently demonstrated that the immune-modulatory effects of ruxolitinib might at least in part be due to an inhibitory effect on dendritic cell (DC) biology (Heine et al., Blood 2014). Dendritic cells are important antigen-presenting cells, which mature and migrate from the periphery into draining lymph nodes upon antigen contact to prime T cells. This process follows chemokine gradients and requires cytoskeletal rearrangement to allow cell movement along the gradients. Members of the family of small GTPases are critically involved in cell chemotaxis, migration and formation of the immunological synapse allowing DC-T cell interaction. The aim of this study was to define in detail the impact of the JAK inhibitor ruxolitinib on DC migration.Methods: CD14+ cells isolated from human buffy coats were differentiated for seven days in the presence of GM-CSF and IL-4 to moDCs and finally matured with LPS. Murine bone marrow-derived DCs (bmDCs) were generated by flushing bone marrow from femura and tibiae of mice and subsequent plating of the cells in medium containing GM-CSF followed by final maturation by LPS. Migration of DCs was analyzed in Transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards CCL19 gradients. Adhesion of cells was tested on different substrates and phenotypic DC marker checked by flow cytometry. Signaling events were analyzed by Western Blot to evaluate changes in phosphorylation levels of relevant proteins of the RhoA/ROCK-pathway.Results: 2D-migration of ruxolitinib-exposed DC on fibronectin is dose-dependently reduced in vitro. By analyzing the migratory phenotype of human moDCs within 3D-collagen gels, ruxolitinib-exposed DCs are still able to sense chemokine gradients as they form lamellipodia at the leading edge of the cell, whereas the retraction of the uropod is clearly inhibited. As a consequence, cell velocity, accumulated and euclidean distance are all significantly reduced by ruxolitinib. These effects are already seen 30 minutes after ruxolitinib exposure. Notably (and in line with the morphological chemokine-sensing by lamellipodia formation), the surface expression of the CCL19-sensing chemokine receptor CCR7 is not altered by ruxolitinib. Moreover, we could not detect any changes of integrin expression or the adhesion of mature DC to fibronectin or collagen as a potential reason for lowered migration due to increased "stickiness" of the DCs. Additional in vivo studies could show that the JAK inhibitor potently reduces homing of bmDCs into draining lymph nodes in mice. Notably, the impaired DC migration is independent of JAK inhibition, as siRNA knockdown experiments revealed that DCs with JAK1, JAK2 or JAK1/2 knockdown migrate appropriately in vitro applying again the 3D-migration system. On a molecular level we could show a reduced phosphorylation of myosin-light chain phosphatase 1 (MYPT1), a direct target of the Rho-associated protein kinase (ROCK) in ruxolitinib-treated moDC upon CCL19 stimulation. Interestingly, the known inhibition of DC activation and maturation seen after treatment with ruxolitinib could not be observed if cells are incubated with the ROCK inhibitor Y-27632. Whereas pharmacological inhibition of ROCK mimicked the migration phenotype seen in moDC exposed to ruxolitinib.Conclusion: RhoA/ROCK pathway is critical for cell migration, as ROCK is a downstream effector of RhoA and leads to stabilization of the actin cytoskeleton via cofilin and acto-myosin II contraction by the myosin light chain. The observed reduction of ROCK activity may reveal an important novel mechanism of ruxolitinib-induced inhibition of DC migration. Our current efforts focus on the validation of ROCK as off-target JAK1/2-independent target kinase of ruxolitinib as potential mediator of the observed DC inhibitory effects, which may at least in part also explain the potential therapeutic impact of ruxolitinib for treatment of GvHD or autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF.

The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells.

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2–dependent adult T-cell leukemia

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.

NK cell modulation by JAK inhibition.

NK cells are innate immune effector cells recognizing and killing virus-infected or malignant cells [1]. NK cell function is tightly regulated by a complex balance between various activating and inhibitory NK cell receptors. In addition, several cytokines (IL-2, IL-12, IL-15 and IL-18) mediated via the JAK/STAT pathway are critical for NK cell development and activation. Thus it is tempting to speculate that the novel class of immune-modulatory compounds (i.e. the JAK inhibitors) are able to modify NK cell biology in vitro and in vivo. We recently support this idea by demonstrating that the first approved JAK1/2/(3) inhibitor ruxolitinib dramatically impacts NK cell biology in vivo in patients suffering from myeleloproliferative neoplasia (MPN)[2]. Ruxolitinib-treated MPN patients have drastically reduced circulating NK cell numbers. Individuals deficient in NK cells due to an inherited genetic disposition (i.e. JAK3 and/or STAT5B mutations that cause severe combined immunodeficiency (SCID) syndrome in humans) are prone to severe infections. Therefore, it is not surprising to see that patients with MPN exposed to the JAK inhibitor, who develop infections, have lower NK cell counts compared to non-infected individuals. However, we also clearly state that severe infections (tuberculosis, progressive multifocal encephalopathy, toxoplasmosis), even though described during ruxolitinib therapy, are relatively infrequent[3]. The most prevalent types of infections during ruxolitinib therapy are urinary tract infection and herpes zoster reactivation, which is known to be linked to NK cell deficiency. However, there are huge differences between patients with inherited disruption of the JAK/STAT pathway as opposed to patients treated with ruxolitinib. Firstly, we could show that the drug-related effect is reversible. In patients who had to stop the therapy due to side effects, NK cell levels rose back to normal values. Furthermore, the main difference is that in patients exposed to the drug, JAK/STAT inhibition lasts only for several hours (3–4) due to the pharmacokinetic properties of the drug. As a consequence of the bi-daily intake of the drug, the total number of JAK/STAT inhibition may last a maximum of 6–8 hours[4]. In between the JAK/STAT pathway is active and allows residual cells to be activated relatively normal. In contrast, for limiting the hyper-inflammation going along with the MPN, this inhibition time appears to be sufficient to drastically reduce symptom burden and shrink the spleen[5]. Furthermore, it also suffices to clearly deplete NK cells in MPN patients. But why is this the case? The increased ratio of immature to mature NK cells during ruxolitinib therapy provides some hints, that the development of mature NK cells is affected, which could be explained by the inhibition of various important cytokine signals essential for NK maturation (i.e. IL-2 and IL-15). Reduced NK cell numbers in ruxolitinib-exposed patients may at least in part be due to defective NK cell maturation, which results in the time-dependent NK cell drop. In line with this idea, data demonstrated that NK cell precursors lacking IL-15Rβ also exhibit a NK cell differentiation defect leading to severe immunodeficiency. Moreover, IL-15R integrates signals via its common γ-chain in committed NK cell precursors and fosters immature to mature NK cell differentiation as well as the cytokine supports mature NK cell survival. Thus, it is tempting to speculate that ruxolitinib interferes with essential cytokine signals for terminal NK cells maturation and explaining the shift to the more immature NK cell phenotype. Strikingly, ruxolitinib not only affects NK cell biology in vivo, but also impairs NK cell function in vitro. Mainly, cytokine-mediated activation is blocked by the drug and most importantly it inhibits NK killing activity, as it interferes amongst others also with the lytic synapse formation process. We are currently investigating in detail the impact of the drug on the migration process of immune cells and early data clearly highlight the effects of the compound on cytoskeletal rearrangement. This may at least in part also affect the potential of NK cells to go into stable contact with target cells, thereby limiting their killing activity. These data may be of particular relevance when considering the application of the drug as cancer-inflammation reverting compound (e.g. in pancreatic cancer[6]) or when applying the drug as immune-suppressant after allogeneic stem cell transplantation[7], as it may also interfere with cancer immune-surveillance or the graft-versus-disease effect in patients thereby fostering disease recurrence. Considering our data, an important question that arises is how long-term ruxolitinib therapy shapes the immune system after years or even decades. The figure depicts the major effects of the JAK inhibitor ruxolitinib on NK cells in vivo (in MPN patients) and in vitro. Figure 1 The figure depicts the major effects of the JAK inhibitor ruxolitinib on NK cells in vivo (in MPN patients) and in vitro

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2-dependent adult T-cell leukemia

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. The HTLV-1-encoded protein Tax up-regulates Bcl-xL expression and constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems resulting in amplified JAK/STAT signaling. Consequently, inhibition of JAK signaling reduces ex vivo proliferation of PBMCs from ATL patients in smoldering and chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in IL-2-dependent and IL-2-independent ATL cell lines. The highly selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen and the Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. An examination of the mechanistic underpinnings of this combination highlighted a stimulation of Bim and PUMA expression and reduced phosphorylation of BAD upon cellular exposure to ruxolitinib. The combination was noted to strongly activate BAX, effect mitochondrial depolarization and increase caspase 3/7 activity that leads to PARP and Mcl-1 cleavage. Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy while the combination dramatically lowered tumor burden and prolonged survival in an aggressive ATL murine model. Critically, this combination strongly blocked ex vivo proliferation of five ATL patients’ PBMCs. These studies provide support for a therapeutic trial in patients with smoldering and chronic ATL using a drug combination that inhibits JAK signaling and anti-apoptotic protein Bcl-xL.

Abstract CT114: A phase II trial of exemestane and ruxolitinib for aI-resistant ER+ breast cancer: Interim safety, efficacy, and biomarker analysis

Abstract Resistance to aromatase inhibitors in ER+ breast cancer leads to recurrence and progression in the metastatic setting. JAK/STAT pathway activation is a resistance mechanism that could potentially be overcome with the use of JAK inhibitor therapy. Methods: We performed a phase II trial of exemestane, 25 mg daily, and ruxolitinib, 25 mg BID, in postmenopausal women with advanced, ER+ breast cancer who had progressed on a non-steroidal aromatase inhibitor and had either measureable or bone-only disease. A Simon 2-stage design was employed. A “go” decision to second stage would occur if fewer than 5/15 patients experienced any grade 3/4 toxicity requiring discontinuation from the study within the first treatment cycle. Results: Fifteen patients were enrolled; during cycle 1, no patient discontinued for toxicity and 1 patient went off study for progression of bone disease. 36 grade 3 events occurred; anemia was most common (n = 5), requiring transfusion in all patients. 47% required dose reduction. No partial or complete responses occurred; 3/15 (20%) had stable disease ≥6 months (clinical benefit, CB). Baseline CRP ≥8 was significantly associated with CB (3/3 CB vs. 1/11 non-CB; p = 0.011); other markers, including baseline ESR, IL-6 genotype status and primary tumor phosphoSTAT3 expression were not associated with CB in this small sample, though high tumoral pSTAT3 was seen in 66% of CB and 33% of non-CB. A novel pharmacodynamic (PD) assay to assess STAT3 phosphorylation in peripheral blood mononuclear cells after ruxolitinib exposure demonstrated differential effects in patients with CB vs. those without CB. Conclusions: The combination of exemestane and the JAK2 inhibitor ruxolitinib met safety criteria for continued enrollment. Anemia, an expected toxicity of R, was common and the high rate of severe anemia and need for dose reductions has led to a decision to reduce the starting dose of ruxolitinib to 15 mg BID moving forward. Promising predictive markers, including CRP, tumor pSTAT3 and a novel PD assay for pSTAT3 will be further evaluated. Citation Format: Angela M. DeMichele, Christopher B. Colameco, Anna Kalota, Andrea B. Troxel, Robin Holmes, Rebecca Cimildoro, Kelly Zafman, Kevin R. Fox, Susan M. Domchek, Keerthi Gogineni, Angela R. Bradbury, Jennifer M. Matro, Natalie Shih, Michael D. Feldman, Amy S. Clark, Elizabeth O. Hexner, Jacqueline F. Bromberg. A phase II trial of exemestane and ruxolitinib for aI-resistant ER+ breast cancer: Interim safety, efficacy, and biomarker analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT114. doi:10.1158/1538-7445.AM2015-CT114

Targeting glutamine metabolism in myeloproliferative neoplasms

JAK2(V617F) mutation can be detected in the majority of myeloproliferative neoplasm (MPN) patients. The JAK2 inhibitor Ruxolitinib is the first FDA-approved treatment for MPNs. However, its use is limited by various dose related toxicities. Here, we studied the metabolic state and glutamine metabolism of BaF3-hEPOR-JAK2V617F and BaF3-hEPOR-JAK2WT cells. We found that the JAK2(V617F)-mutant cells were associated with increased oxygen consumption rate and extracellular acidification rate than the JAK2(WT) cells and there was an increased glutamine metabolism in JAK2(V617F)-mutant cells compared to wild-type cells. Glutaminase (GLS), the key enzyme in glutamine metabolism, was upregulated in the JAK2(V617F)-mutant BaF3 cells compared to the JAK2(WT) BaF3 cells. In MPN patient peripheral blood CD34+ cells, GLS expression was increased in JAK2(V617F)-mutant progenitor cells compared to JAK2 wild-type progenitor cells from the same patients and GLS levels were increased at the time of disease progression compared to at earlier time points. Moreover, GLS inhibitor increased the growth inhibitory effect of Ruxolitinib in both JAK2(V617F)-mutant cell lines and peripheral blood CD34+ cells from MPN patients. Therefore, GLS inhibitor should be further explored to enhance the therapeutic effectiveness of JAK2 inhibitor and allow the administration of lower doses of the drug to avoid its toxicity.

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.

Myeloproliferative Neoplasms: A Contemporary Review.

Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) constitute the BCR-ABL1-negative myeloproliferative neoplasms and are characterized by mutually exclusive Janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) mutations; respective frequencies of these mutations are approximately 95%, 0%, and 0% in PV, 60%, 20%, and 3% in ET, and 60%, 25%, and 7% in PMF. These mutations might be accompanied by other mutations that are less specific to myeloproliferative neoplasms but are prognostically relevant, such as additional sex combs-like 1 (ASXL1). Characteristic bone marrow morphology is required for World Health Organization-compliant diagnosis, especially in distinguishing ET from prefibrotic PMF and masked PV. Survival is the longest in ET, although still inferior to that of the age- and sex-matched control population; median survivals for patients younger than 60 years are approximately 33 years for ET, 24 for PV, and 15 for PMF. Major disease complications include thrombosis and leukemic or fibrotic transformation. In PV and ET, risk factors for survival include older age, leukocytosis, and thrombosis, whereas JAK2 mutation in ET is associated with increased risk of thrombosis. In PMF, type 1 or type 1-like CALR mutations are associated with superior and ASXL1 with inferior survival. Prevention of thrombosis in PV is secured by phlebotomy (hematocrit target &lt;45%) and in both PV and ET by low-dose aspirin therapy; high-risk patients derive additional antithrombotic benefit from cytoreductive therapy with hydroxyurea as first-line and interferon-alfa and busulfan as second-line drugs of choice. Although the JAK inhibitor ruxolitinib was recently approved for use in hydroxyurea-resistant PV, its role in routine clinical practice remains debatable. In myelofibrosis, stem cell transplant is the current treatment of choice for genetically or clinically high-risk disease; for all other patients requiring treatment, participation in clinical trials may be preferred because currently available drugs, including JAK inhibitors, are palliative and not shown to be disease modifying.