JAK Inhibitor Ruxolitinib Research Articles

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

A novel silicone derivative of natural osalmid (DCZ0858) induces apoptosis and cell cycle arrest in diffuse large B-cell lymphoma via the JAK2/STAT3 pathway

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignant tumor characterized by diffuse growth. DCZ0858 is a novel small molecule with excellent antitumor effects in DLBCL. This study explored in depth the inhibitory effect of DCZ0858 on DLBCL cell lines. Cell Counting Kit-8 (CCK-8) and plate colony formation assays were used to evaluate cell proliferation levels. Flow cytometry was employed to analyze apoptosis and the cell cycle, and western blotting was used to quantify the expression of cell cycle regulators. The results indicated that DCZ0858 inhibited cell growth in a concentration-dependent and time-dependent manner while inducing no significant toxicity in normal cells. Moreover, DCZ0858 initiated cell apoptosis via both internal and external apoptotic pathways. DCZ0858 also induced cell cycle arrest in the G0/G1 phase, thereby controlling cell proliferation. Further investigation of the molecular mechanism showed that the JAK2/STAT3 pathway was involved in the DCZ0858-mediated antitumor effects and that JAK2 was the key target for DCZ0858 treatment. Knockdown of JAK2 partly weakened the DCZ0858-mediated antitumor effect in DLBCL cells, while JAK2 overexpression strengthened the effect of DCZ0858 in DLBCL cells. Moreover, a similar antitumor effect was observed for DCZ0858 and the JAK2 inhibitor ruxolitinib, and combining the two could significantly enhance cancer-suppressive signaling. Tumor xenograft models showed that DCZ0858 inhibited tumor growth in vivo and had low toxicity in important organs, findings that were consistent with the in vitro data. In summary, DCZ0858 is a promising drug for the treatment of DLBCL.

IL-6 produced by prostate epithelial cells stimulated with Trichomonas vaginalispromotes proliferation of prostate cancer cells by inducing M2 polarization of THP-1-derived macrophages.

Trichomonas vaginalis (Tv), a protozoan parasite causing sexually-transmitted disease, has been detected in tissue of prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). IL-6, a mediator of chronic inflammation, induces the progression of prostate cancer, and influences the polarization of M2 macrophages, which are the main tumor-associated macrophages. We investigated whether IL-6 produced by human prostate epithelial cells stimulated with Tv induces the M2 polarization of THP-1-derived macrophages, which in turn promotes the progression of PCa. Conditioned medium was prepared from Tv-infected (TCM) and uninfected (CM) prostate epithelial cells (RWPE-1). Thereafter conditioned medium was prepared from macrophages after incubation with CM (M-CM) or TCM (M-TCM). RWPE-1 cells infected with Tv produced IL-6 and chemokines such as CCL2 and CXCL8. When human macrophages were treated with conditioned medium of RWPE-1 cells co-cultured with Tv (TCM), they became polarized to M2-like macrophages as indicated by the production of IL-10 and TGF-β, and the expression of CD36 and arginase-1, which are M2 macrophage markers. Moreover, proliferation of the M2-like macrophages was also increased by TCM. Blockade of IL-6 signaling with IL-6 receptor antibody and JAK inhibitor (Ruxolitinib) inhibited M2 polarization of THP-1-derived macrophages and proliferation of the macrophages. To assess the effect of crosstalk between macrophages and prostate epithelial cells inflamed by Tv infection on the growth of prostate cancer (PCa) cells, PC3, DU145 and LNCaP cells were treated with conditioned medium from THP-1-derived macrophages stimulated with TCM (M-TCM). Proliferation and migration of the PCa cells were significantly increased by the M-TCM. Our findings suggest that IL-6 produced in response to Tv infection of the prostate has an important effect on the tumor microenvironment by promoting progression of PCa cells following induction of M2 macrophage polarization.

Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

STAT3/5 Inhibitors Suppress Proliferation in Bladder Cancer and Enhance Oncolytic Adenovirus Therapy.

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.

MiR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

Myeloproliferative and lymphoproliferative disorders: State of the art.

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal disorders complicated mainly by vascular events and transformation to myelofibrosis (for PV and ET) or leukemia. Although secondary malignancies, in particular, lymphoproliferative disorders (LPNs), are rare, they occur at a higher frequency than found in the general population, and there has been recent scientific discussion regarding a hypothetical relationship between treatment with JAK inhibitors in MPN and the risk of development of LPN. This has prompted increased interest regarding the coexistence of MPN and LPN. This review focuses on the role of JAK2 and the JAK/STAT pathway in MPN and LPN, whether there is a role for the genetic background in the occurrence of both MPN and LPN and whether there is a role for cytoreductive drugs in the occurrence of both MPN and LPN. Furthermore, whether an increased risk of lymphoma development is limited to patients who receive the JAK inhibitor ruxolitinib, is a more general phenomenon that occurs following JAK1/2 inhibition or is associated with preferential JAK1 or JAK2 targeting is discussed.

Dysregulated IL-6/GP130/JAK Signaling in Calreticulin Mutated Myeloproliferative Neoplasms (MPN)

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

Ruxolitinib Reverses Checkpoint Inhibition By Downregulating PD-L1 and PD-L2 Expression on Both Tumor and Stromal Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) tumor cells evade host immunity through the interaction of PD-L1 and PD-L2 to PD-1 on T-cells. This creates an immunosuppressive milieu in the bone marrow (BM) microenvironment. The immune inhibitory proteins PD-L1 and PD-L2 are highly expressed in MM BM. Moreover, increased expression of these proteins are associated with resistance to treatment in MM. Ruxolitinib (RUX) is a JAK1/2 inhibitor that is effective for the treatment of myeloproliferative diseases. In this study, we examined PD-L1 and PD-L2 gene and protein expression in the BM of MM patients with progressive disease (PD) or in complete remission (CR). We further investigated the effects of RUX on expression of PD-L1 and PD-L2 in MMBM, and the effect of RUX in combination with anti-MM agents in vitro and in vivo. Material and Methods: BM mononuclear cells (MCs) and serum were collected from MM patients and healthy subjects after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the mice. The cells were cultured and treated with or without RUX and then were determined by qPCR, flow cytometric analysis, ELISA, and western blot. Results and Discussion: The results from qPCR and flow cytometric assays showed that PD-L1 and PD-L2 gene expression was markedly increased in BMMCs from MM patients with PD compared with patients in CR or with healthy controls. We further investigated the effects of RUX on PD-L1 and PD-L2 expression in primary and stromal cells from MM patients' BM samples in vitro. RUX treatment markedly reduced PD-L1and PD-L2 gene and protein expression in the MM tumor cells cultured alone or co-cultured with stromal cells in a concentration dependent pattern. We then determined whether RUX can augment the anti-MM effects of T-cells in vitro. RUX (0, 0.1, 0.5, 1, and 5 µM) increased MM cell apoptosis in the presence of IL-2 stimulated T-cells in a concentration dependent fashion, to a similar degree to anti-PD-1 (0, 0.5, 1, 5, and 10 µg/ml) or anti-PD-L1 (0, 0.5, 1, 5, and 10 µg/ml) antibody treatment. Moreover, the combination of RUX with anti-PD-1 or anti-PD-L1 antibody increased T-cell-induced MM cell apoptosis more than the agents alone. To evaluate the efficacy of drugs in vivo, severe combined immune deficient mice implanted with the human MM xenograft LAGκ-2 were treated with RUX (30mg/kg). The results showed PD-L1 expression in the xenograft was significantly decreased in RUX-treated mice compared with the untreated control group. In contrast, RUX had no effect on PD-1 expression on T-cells. Conclusion: The PD-L1/PD-1 pathway delivers inhibitory signals that regulate both peripheral and central tolerance, and inhibit anti-tumor immune-mediated responses. This study demonstrated that the JAK inhibitor RUX downregulated PD-L1 and PD-L2 expression in both MM tumor and stromal cells. We also demonstrated that RUX alone increased T-cell-induced apoptosis of MM cells; and, moreover, the combination of RUX with anti-PD-1 and anti-PD-L1 further increased apoptosis. The results suggest that JAK inhibitors may be effective for treating MM patients through their ability to reduce expression of checkpoint proteins involved in the development of immune resistance. Thus, JAK inhibitors should help overcome the immune resistance generated by these proteins for patients with this B-cell malignancy. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Incyte Corporation.: Consultancy, Research Funding; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; Incyte Corporation.: Consultancy, Research Funding; Janssen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Amag: Consultancy, Speakers Bureau.

Evaluation of Virus-Specific T Cell Responses in Patients Affected with Myeloproliferative Neoplasms Treated with Ruxolitinib

Background The classic Ph-negative myeloproliferative neoplasms (MPN) are a group of clonal haematopoietic disorders, including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) (either primary or secondary), that share the deregulation of JAK-STAT signalling. Over the past few years, there have been significant changes in the therapeutic landscape of MPN, thanks to the approval of the JAK-inhibitor ruxolitinib. Despite its efficacy and beyond its well described haematological toxicity, the drug may also cause an increased risk of reactivation of silent infections (e.g., tuberculosis, hepatitis B virus and varicella zoster virus). Less is known regarding other opportunistic viral pathogens, such as human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The aim of this study was to evaluate the viral load and T cell responses to HCMV and EBV in 25 MPN patients (6 PV, 5 ET, 14 MF) treated with ruxolitinib. Methods Peripheral blood was collected monthly for viral genome quantification using real-time PCR (EBV-DNA and HCMV-DNA) and determination of T cell subsets by flow cytometry (absolute number of CD3+, CD3+CD4+, and CD3+CD8+). The T cell responses specific to HCMV and EBV were evaluated monthly using IFN-γ ELISPOT assay. Results were normalized to CD4+ and CD8+ T cell count. Correlations between T cell responses were evaluated by regression models for panel data (with random or fixed effects based on result of Hausman test). P-values <0.05 were considered significant. Results Of 25 patients treated with Ruxolitinib (median time of treatment: 5 years, range: 0.2-10.5 years), 15 (60%) had CD4+ T cells and 7 (28%) had CD8+ T cells below normal ranges. The reduction was observed in all MPN subtypes: CD4+ T cells were reduced in 50% of PV, 80% of ET, 57% of MF; CD8+ T cells were reduced in 33% of PV, 60% of ET and 14% of MF. The reduction was not different based on disease duration (<10 years vs >= 10 years since diagnosis) while it correlated with duration of ruxolitinib treatment: patients receiving ruxolitinib for more than 5 years had more frequently a reduction of CD4+ T cells and/or CD8+ T cells compared with patients treated for less than 5 years (93% vs 45% P 0.021). Moreover, the median number of CD4+ cells and CD8+ cells were lower in patients treated >= 5 years vs <5 years (366 vs 558 cells/μl P = 0.043 and 206 vs 365 cells/μl P = 0.002). During the study, reactivation of EBV was observed in 76% of patients while only 8% experienced reactivations of HCMV ; only one patient (4%) experienced Varicella Zoster Virus clinical reactivation. Both the EBV-specific and CMV-specific CD4+ and CD8+ T cell responses showed an inverse correlation with detectable EBV DNA and HCMV DNA although it was not statistically significant (all p-values>0.2). When normalizing results to CD4 and CD8 T cell counts we observed again an inverse correlation without statistical significance (all p-values>0.1). Both the EBV-specific and CMV-specific CD4+ and CD8+ T cell responses had an inverse correlation with the dosage of ruxolitinib (<= 20 mg/die vs > 20 mg/die) although not statistically significant (all p-values>0.06). Conclusions Our study suggests that a reduction of CD4+ T cells and CD8+ T cells is frequently observed in MPN patients treated with ruxolitinib and is associated with treatment duration (>= 5 years vs <5 years). Reactivation of EBV occurs frequently while reactivation of CMV is less frequent. As the virus-specific T cell responses seem to have a trend to an inverse correlation with detectable EBV DNA and HCMV DNA we hypothesize that an impaired response makes the patient unable to clear the virus. Moreover, the virus-specific T cell responses seem to have a trend to an inverse correlation with ruxolitinib dosage, thus pointing to a potential role of the drug in favouring viral reactivation. To corroborate this hypothesis, virus-specific T cell responses need to be analysed in a larger number of patients with MPN under ruxolitinib treatment. Disclosures Rumi: novartis: Honoraria, Research Funding. Arcaini:Gilead Sciences: Research Funding; Celgene: Speakers Bureau; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses.

CRLF2 rearrangement in Ph-like acute lymphoblastic leukemia predicts relative glucocorticoid resistance that is overcome with MEK or Akt inhibition.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

Mechanisms of action of ruxolitinib in murine models of hemophagocytic lymphohistiocytosis

AbstractHemophagocytic lymphohistiocytosis (HLH) is an often-fatal disorder characterized by the overactivation of T cells and macrophages that excessively produce proinflammatory cytokines, including interferon-γ (IFN-γ). Previously, we reported that the JAK inhibitor ruxolitinib dampens T-cell activation and lessens inflammation in a model of HLH in which perforin-deficient (Prf1−/−) mice are infected with lymphocytic choriomeningitis virus (LCMV). Ruxolitinib inhibits signaling downstream of IFN-γ, as well as several other JAK-dependent cytokines. As a consequence, it remained unclear whether ruxolitinib was exerting its beneficial effects in HLH by inhibiting IFN-γ signaling or by targeting signaling initiated by other proinflammatory cytokines. To address this question, we compared the effects of ruxolitinib with those obtained using an IFN-γ–neutralizing antibody (αIFN-γ) in 2 murine HLH models. In both models, ruxolitinib and αIFN-γ reduced inflammation-associated anemia, indicating that ruxolitinib operates in an IFN-γ–dependent manner to reverse this HLH manifestation. In contrast, the number and activation status of T cells and neutrophils, as well as their infiltration into tissues, were significantly reduced following treatment with ruxolitinib, but they remained unchanged or were increased following treatment with αIFN-γ. Notably, despite discontinuation of ruxolitinib, LCMV-infected Prf1−/− mice exhibited enhanced survival compared with mice in which αIFN-γ was discontinued. This protective effect could be mimicked by transient treatment with αIFN-γ and a neutrophil-depleting antibody. Thus, ruxolitinib operates through IFN-γ–dependent and -independent mechanisms to dampen HLH by targeting the deleterious effects of T cells and neutrophils, with the latter representing an unappreciated and understudied cell type that contributes to HLH pathogenesis.

Phenotype-based drug screening reveals association between venetoclax response and differentiation stage in acute myeloid leukemia.

Ex vivo drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia. However, accurate blast-specific drug responses cannot be measured with homogeneous "add-mix-measure" cell viability assays. In this study, we implemented a flow cytometry-based approach to simultaneously evaluate the ex vivo sensitivity of different cell populations in 34 primary acute myeloid leukemia samples to seven drugs and 27 rational drug combinations. Our data demonstrate that different cell populations present in acute myeloid leukemia samples have distinct sensitivity to targeted therapies. Particularly, blast cells of FAB M0/1 acute myeloid leukemia showed high sensitivity to venetoclax. In contrast, differentiated monocytic cells abundantly present in M4/5 subtypes showed resistance to Bcl-2 inhibition, whereas immature blasts in the same samples were sensitive, highlighting the importance of blast-specific readouts. Accordingly, in the total mononuclear cell fraction the highest BCL2/MCL1 gene expression ratio was observed in M0/1 and the lowest in M4/5 acute myeloid leukemia. Of the seven tested drugs, venetoclax had the highest blast-specific toxicity, and combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib effectively targeted all venetoclax-resistant blasts. In conclusion, we show that ex vivo efficacy of targeted agents and particularly Bcl-2 inhibitor venetoclax is influenced by the cell type, and accurate blast-specific drug responses can be assessed with a flow cytometry-based approach.

Abstract 3836: Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML

Abstract Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p < 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of < 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate > 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values < 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3836.

Abstract 1287: Targeting pleckstrin-2 for the JAK2-STAT and PI3K-Akt pathways in cancer therapy

Abstract Pleckstrin-2 (Plek2) is a widely expressed PH domain containing protein that binds to phosphoinositide with unclear functions. Our published reports reveal that Plek2 is overexpressed in JAK2 V617F mutation positive myeloproliferative neoplasms (MPNs). We identified that Plek2 is a downstream effector of the JAK2-STAT5 pathway. Furthermore, through a mouse genetic approach, we discovered that knockout of Plek2 significantly ameliorated the MPN phenotypes and reverted lethality and thrombosis in JAK2 V617F knockin mice. These studies demonstrate that Plek2 is critical for the pathogenesis of MPNs with the activated JAK2-STAT5 pathway, and form a strong foundation for the development of Plek2 inhibitors for the treatment of MPNs. Importantly, our published study shows that Plek2 knockout mice do not develop anemia or cytopenia, indicating Plek2’s oncogenic potential is only in the disease background, which makes Plek2 inhibitors less likely to cause severe side effects compared to JAK inhibitor ruxolitinib. Based on these studies, we used an in silico approach to screen for putative Plek2 binding small molecules and identified hit compounds that bind to the DEP domain of Plek2. Further medicinal chemistry studies identified lead compound NUP-17d that inhibited proliferation of the hyperproliferative hematopoietic cells with potency comparable to ruxolitinib. In addition, NUP-17d also blocked proliferation of several Plek2-overexpressing solid tumor cell lines. Our biochemical assays showed that NUP-17d inhibited Akt phosphorylation. Further mechanistic studies revealed that Plek2 functions as a central hub to mediate the JAK2-STAT and PI3K-Akt pathways to promote tumor cell proliferation. Specifically, Plek2 binds to and recruits PI3K-produced PtdIns(3,4)P2 and PI3K effector proteins including Akt, PDK1, PDK2, and mTOR, which forms a complex and significantly enhances the PI3K signaling. In addition to the biochemical assays, we also demonstrated Plek2’s function through a mouse genetic approach using a Pten hematopoietic specific knockout mouse model. Knockout of Plek2 significantly reverted the myeloproliferative phenotype in these mice and markedly extended their survival. Therefore, NUP-17d blocks tumor cell proliferation through the disruption of the Plek2 complex and inhibition of the PI3K-Akt pathway. Indeed, treatment of an erythropoietin-induced myeloproliferative mouse model with NUP-17d also demonstrated its potent inhibitory effect in myeloproliferation in vivo. These study establishes Plek2 as a oncoprotein mediating JAK2-STAT and PI3K-Akt signaling pathways. In addition to hematologic malignancies, Plek2 is also found to be highly upregulated with an associated worse prognosis in many solid tumors (oncomine, kmplot). Therefore, our Plek2 inhibitors will have a broad impact in cancer therapy, especially cancers with upregulated JAK2-STAT or PI3K-Akt pathway. Citation Format: Xu Han, Yang Mei, Gary E. Schiltz, Rama K. Mishra, Atul D. Jain, Peng Ji. Targeting pleckstrin-2 for the JAK2-STAT and PI3K-Akt pathways in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1287.

The Rationale for Immunotherapy in Myeloproliferative Neoplasms.

The classic, chronic Philadelphia chromosome negative myeloproliferative neoplasms (MPN)-essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)-are clonal malignancies of hematopoietic stem cells and are associated with myeloproliferation, organomegaly, and constitutional symptoms. Expanding knowledge that chronic inflammation and a dysregulated immune system are central to the pathogenesis and progression of MPNs serves as a driving force for the development of agents affecting the immune system as therapy for MPN. This review describes the rationale and potential impact of anti-inflammatory, immunomodulatory, and targeted agents in MPNs. The advances in molecular insights, especially the discovery of the Janus kinase 2 (JAK2) V617F mutation and its role in JAK-STAT pathway dysregulation, led to the development of the JAK inhibitor ruxolitinib, which currently represents the cornerstone of medical therapy in MF and hydroxyurea-resistant/intolerant PV. However, there remain significant unmet needs in the treatment of these patients, and many agents continue to be investigated. Novel, more selective JAK inhibitors might offer reduced myelosuppression or even improvement of blood counts. The recent approval of a novel, long-acting interferon for PV patients in Europe, might eventually lead to its broader clinical use in all MPNs. Targeted immunotherapy involving monoclonal antibodies, checkpoint inhibitors, or therapeutic vaccines against selected MPN epitopes could further enhance tumor-specific immune responses. Immunotherapeutic approaches are expanding and hopefully will extend the therapeutic armamentarium in patients with myeloproliferative neoplasms.

Engineered Bcor mutations lead to acute leukemia of progenitor B-1 lymphocyte origin in a sensitized background

AbstractApproximately 10% of NUP98-PHF23 (NP23) mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor (Bcor) gene, most commonly within a 9-bp “hotspot” in Bcor exon 8. To determine whether experimentally engineered Bcor mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats–associated protein 9 to introduce a Bcor frameshift mutation into NP23 hematopoietic stem and progenitor cells through the use of Bcor small guide RNAs (Bcor sgRNAs). Recipient mice transplanted with NP23 bone marrow or fetal liver cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that Bcor mutations collaborate with NP23 to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.

PS1439 ENHANCED SUPPORT OF MYELOFIBROSIS STEM CELLS IN NEXT GENERATION HUMANIZED MICE

Background:Myelofibrosis (MF) is a hematopoietic stem cell (HSC) neoplasm characterized by an expansion of myeloid cells followed by deposition of fibers in the bone marrow (BM) and leukemic transformation depending on the clonal evolution of MF stem cells (MF SCs). Mutations in the receptor tyrosine kinase JAK2, the endoplasmic reticulum chaperone CALR or the thrombopoietin receptor (MPL) drive the disease phenotype through cytokine‐independent activation of the JAK‐STAT signaling pathway. Patient‐derived xenograft (PDX) models have emerged as powerful tools for investigating aggressive malignancies, such as acute leukemia's. However, the development of less‐aggressive malignancies, like MF, is often limited in PDX models. We hypothesized that the constitutive expression of human cytokines and growth factors in immunocompromised mice (i.e. MISTRG mice) may provide a supportive microenvironment for MF SC development.Aims:To develop a pre‐clinical MF PDX model that faithfully recapitulates the disease phenotype and the genetic heterogeneity observed in patients.Methods:Purified peripheral blood (PB) stem and progenitor (CD34+) cells from 14 MF patients were transplanted intra‐hepatically into sub‐lethally irradiated newborn MISTRG mice and NSG mice (controls). For secondary transplantations, human CD45+ cells purified from primary animals were transplanted intra‐hepatically. Mice were sacrificed 5–26 weeks (median: 9.8 weeks) post‐transplantation and characterized by flow cytometry, immunohistochemistry, and mutational profiling.Results:MISTRG mice supported significantly higher human MF engraftment in the BM (29.20% vs 4.175%, p < 0.0001), the PB (48.70% vs. 0.73%, p < 0.0001) and the spleen (8.19% vs. 0.33%, p < 0.0001) compared to NSG mice. All 14 investigated patient samples engrafted in MISTRG mice, while 9/14 patient samples engrafted in NSG mice. MISTRG mice exhibited superior engraftment independent of risk categories (DIPSS, MIPSS70, and MYSEC), disease stage (chronic, accelerated) and diagnoses (primary, secondary MF). Both NSG and MISTRG mice supported robust monocytic and granulocytic engraftment in the BM. Furthermore, immunohistochemistry revealed human megakaryocytes in both strains. An increase in reticulin fibers was detected in MISTRG mice transplanted with 1 patient sample that was analyzed after 18 weeks. To determine whether the engrafted human cells were derived from the MF clone, NSG and MISTRG mice that showed >10% human engraftment were analyzed for driver mutations (JAK2 or CALR). JAK2 or CALR mutations were preserved in all NSG (7/7) and MISTRG (39/39) mice transplanted with the respective sample. Next, we performed targeted sequencing of 54 myeloid genes frequently mutated in myeloid neoplasms on 7 NSG mice transplanted with 4 patient samples and 22 MISTRG mice transplanted with 8 patient samples to determine whether the mutational profile of the primary sample was preserved in the corresponding engrafted xenografts. This data showed maintenance of the primary patient sample clonal composition in both NSG and MISTRG mice. Finally, purified human MF cells isolated from primary mice showed myeloid reconstitution in secondary recipients.Summary/Conclusion:These results show that MISTRG mice support robust engraftment of MF SCs across all disease stages and categories and faithfully reproduce the genetic complexity observed in patients. This novel MISTRG MF PDX model is currently being used to assess the effect of the JAK inhibitor (JAKi) Ruxolitinib on engrafted human MF cells.

160 Mechanisms governing promoter methylation and apoptosis in cutaneous T-cell lymphoma: implications for therapy with methotrexate and JAK-inhibitors

Cutaneous T-cell lymphoma (CTCL) is characterized by resistance to apoptosis involving dysregulation of the FAS ligand (FASL) - FAS death receptor pathway. CTCL cells also exhibit constitutively activated STAT proteins, which are known to upregulate and bind DNA methyltransferases (DNMTs). We hypothesized that, like many other tumor suppressor genes, FAS and FASL expression is at least partially regulated by promoter methylation. Earlier, we showed that methotrexate (MTX), a known folate antagonist, acts as a DNA methylation inhibitor via depleting the methyl donor, S-adenosylmethionine (SAM), in CTCL cells. Pyrosequencing analysis of FAS and FASL promoters showed that MTX inhibited methylation of certain CpG sites in these genes, which resulted in increased FAS/FASL expression and subsequent apoptosis via activated caspase 8. We found that shRNA mediated genetic knockdown of DNMT -1 and -3A also resulted in upregulation of FASL mRNA and protein in CTCL cell lines. However, the reduction in FASL promoter methylation induced by MTX was generally superior to that induced by shRNA knockdown and was not improved by a combination of MTX plus knockdown. Because STAT proteins help regulate DNMTs, we also evaluated the effect of multiple JAK inhibitors (fedratinib, tofacitinib, and ruxolitinib) in CTCL cells and Sezary patient’s blood. Among these, fedratinib treatment was most effective in decreasing viability, spheroid formation, and inducing apoptosis. These responses were accompanied by decreased phosphorylation of STAT3 at tyrosine-705. Taken together, our data demonstrate that enhanced apoptosis of CTCL can be induced by MTX, DNMT knockdown and JAK inhibitors. This is consistent with a mechanistic cascade in which activated STATs enhance DNMT activities that in turn suppress the expression of apoptotic factors and promote CTCL survival. Our findings suggest a therapeutic role for MTX and JAK inhibitors in the management of CTCL.