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Chronic myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed bulk transcriptome profiling accompanied by single cell RNA-sequencing on CD34+ stem/progenitor cells from serial patient samples obtained at the chronic MPN and sAML phases, and identified aberrantly increased expression of dual-specificity phosphatase 6 (DUSP6) underlying disease transformation. Genetic and pharmacologic targeting of DUSP6 led to inhibition of S6 and JAK/STAT signaling, resulting in potent suppression of cell proliferation, while also reducing inflammatory cytokine production in primary samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells. Mechanistically, DUSP6 perturbation dampened S6 signaling via inhibition of RSK1, which we identified as a second indispensable candidate associated with poor clinical outcome. Lastly, DUSP6 inhibition potently suppressed disease development across Jak2 V617F and MPL W515L MPN mouse models, and sAML patient-derived xenografts. These findings underscore DUSP6 in driving disease transformation and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. Significance: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945

Myeloproliferative neoplasms (MPNs) are clonally derived from hematopoietic stem/progenitor cells (HSPCs) and typically harbor somatic mutations in one of three genes (JAK2, CALR, MPL) leading to aberrant activation of JAK-STAT signaling. While small molecule inhibitors of JAK2 provide symptomatic benefit for MPN patients, they do not eradicate the underlying malignant clone, nor do they prevent disease progression. Chronic MPNs exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. To understand alterations to the transcriptional landscape underlying MPN disease progression, we performed bulk transcriptome profiling on myelofibrosis (MF) and sAML patient CD34+ HSPCs. Differential gene expression analysis revealed upregulation of dual-specificity phosphatase 6 (DUSP6), which encodes a MAPK phosphatase that regulates ERK signaling, in sAML CD34+ cells. Elevated DUSP6 protein expression accompanying disease progression was confirmed via MPN patient bone marrow immunofluorescence and imaging mass cytometry analysis. We performed further single cell RNA sequencing (scRNA-seq) in conjunction with TotalSeq surface protein marker detection on more than 50,000 sorted CD34+ cells of serial samples from three patients at chronic MPN and sAML stages, and two healthy controls, which revealed DUSP6 among the top 21 genes elevated in all three paired samples across disease progression. Subsequent differentiation trajectory pseudotime analysis demonstrated concomitant elevation of DUSP6 across state trajectories and disease progression. Genetic and pharmacologic targeting of DUSP6 followed by biochemical and mass cytometry analysis identified signaling inhibition through S6 and JAK/STAT, establishing them as novel, non-canonical effectors of DUSP6. DUSP6 inhibition also led to potent suppression of cell proliferation, induction of apoptosis and cell cycle arrest, and reduction of inflammatory cytokine production in primary MPN samples. Furthermore, ectopic DUSP6 expression augmented proliferation and mediated JAK2 inhibitor resistance, while DUSP6 inhibition reduced colony-forming potential of JAK2 inhibitor-persistent patient cells. Mechanistically, DUSP6 suppression dampened S6 signaling via inhibition of RSK1 (RPS6KA1), which we identified as a second indispensable candidate associated with poor clinical outcome via Kaplan-Meier overall survival (Log-rank p = 0.0005) and multivariate (RPS6KA1 expression hazard ratio = 1.60, 95% confidence interval: 1.10, 2.34) analyses of the TCGA LAML cohort. Strong correlation was observed (r = 0.68; p = 0.0009) between RPS6KA1 and DUSP6 expression in CD34+ HSPCs, and pharmacologic inhibition of RSK1 with BI-D1870 suppressed proliferation and colony formation across AML cell lines and primary samples. DUSP6 inhibition in vivo via small molecule inhibitor BCI resolved pathologically elevated hematocrit and white blood cell counts and reduced splenomegaly in Jak2 V617F knock-in mice. In the MPL W515L retroviral transplant model, BCI suppressed leukocytosis while reducing reticulin fibrosis and prolonging survival. In patient-derived xenograft (PDX) model of NSGS mice engrafted with sAML patient CD34+ cells, BCI treatment or DUSP6 knockdown reduced peripheral blood hCD45+ engraftment. Importantly, BCI treatment did not pathologically cause cytopenias or decrease spleen weights in wild-type mice, nor did it reduce hCD45+ engraftment in NSGS PDX mice engrafted with healthy donor CD34+ cells. Lastly, NSGS PDX mice engrafted with MF patient CD34+ cells ectopically expressing DUSP6 demonstrated marked leukocytosis, splenomegaly, and early lethality. These findings underscore DUSP6 in driving MPN disease progression and therapeutic resistance, and highlight the DUSP6-RSK1 axis as a novel, druggable pathway in myeloid malignancies. Figure 1 Figure 1. Disclosures Oh: Abbvie: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Celgene Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Membership on an entity's Board of Directors or advisory committees; PharamaEssentia: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees.

Myelofibrosis (MF) is the deadliest subtype of myeloproliferative neoplasm (MPN) with a median survival of approximately 5 years. Ruxolitinib, a front line therapy for JAK2V617F mutant MPN, can alleviate symptoms of the disease, but does not eliminate the malignant clone and has minimal impact on BM fibrosis and overall survival. Current mouse models do not recapitulate the clinical heterogeneity, clonal genetic composition, or morphological features of MF. Most notably, these models do not generate robust reticulin fibrosis in the bone marrow, the most significant MF pathology. This lack of clinically relevant MF models presents a major barrier to deciphering the complex genetic drivers of the disease and developing effective therapies against it. We evaluated the ability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from MF patients (that contain the MPN-disease initiating population) to give rise to MF in xenotransplanted NSGS mice. >5x104 FACS-sorted CD34+ HSPCs from the peripheral blood of MF patients with JAK2V617F (n=12), CALRindels (n=7) and MPLW515L (n=2) were transplanted into sublethally irradiated (200rads) NSGS mice via X-ray guided intra-tibial injection. We observed robust engraftment of patient-derived cells at 12 weeks post-transplant regardless of their genetic background or donor patient disease severity (Fig 1A). Post-transplant, BM analysis revealed robust expansion of phenotypically defined MF HSCs relative to cord blood CD34+ control recipients, suggesting a permissive niche for MF HSCs to undergo self-renewal. Remarkably, transplantation of CD34+ cells produced other hallmarks of MF in recipient animals such as splenomegaly, thrombocytosis and most importantly BM reticulin fibrosis in all recipients (Fig 1B). We assessed the clonal architecture of engrafted human cells compared to the primary disease in the donor patients through exome sequencing of CD34+ cells prior to transplantation and hCD45+ cells from MF xenografts. We found that the clonal and subclonal mutational landscape observed in CD34+ cells prior to transplantation was maintained in recipient mice (Fig 1C), suggesting that the PDX model accurately reflects the cellular composition of the primary disease. Intriguingly, in two of the xenografted patient samples, we identified additional mutations that were not detected in the primary patient samples using standard sequencing - namely TP53R248Q and EZH2Y663H respectively. Two years after we detected these mutations in PDXs, these MF patients transformed into sAML with acquisition of TP53R248Q and EZH2Y663H mutations. We performed droplet digital PCR and demonstrated that indeed rare pre-leukemic subclones containing these mutations were present at low levels (< 0.01% VAF) in chronic stage MF patients at least two years prior to sAML progression. These data also suggested that these rare subclones responsible for leukemic transformation expand significantly (>300 fold) under the selective pressure of transplantation in NSGS mice. Additional validation of these findings in a further six pre-sAML MF patient samples is currently ongoing. If successful, this model could be used to prospectively identify rising leukemic clones in chronic stage MF patients, which are below the level of detect of standard sequencing as a mechanism to stratify such patients for more aggressive treatments. While sequencing can identify ultra-rare variants, it cannot discern their functional potential for sAML transformation, which is the advantage of this approach. Finally, we harnessed this system for pre-clinical studies, initially focusing on inhibiting the JAK/STAT signaling pathway. Ruxolitinib treatment in MF PDXs produced remarkably similar phenotypes as observed in patients. We observed a small, but significant reduction in engraftment of MF cells in the BM and a sharp reduction in spleen size in Ruxolitinib-treated group compared to vehicle control. Ruxoltinib treatment however did not reduce the frequency of MF HSCs, the disease initiating population or lessen the degree of reticulin fibrosis. These data suggest that this system can be used as a reliable, clinically-relevant drug screening platform. Taken together, we offer the field a critical, previously missing biologically relevant screening system for validation of MPN drug targets identified in cell lines or genetic mouse models prior to moving forward into clinical trials. Disclosures Oh: Blueprint Medicines: Consultancy; Celgene/BMS: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Disc Medicine: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy; Kartos Therapeutics: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy.

The distinct clinical features of myelofibrosis (MF) have been attributed in part to dysregulated inflammatory cytokine production. Circulating cytokine levels are elevated in MF patients; a subset of which have been shown to be poor prognostic indicators. In this study, cytokine overproduction was examined in MF patient plasma and in MF blood cells ex vivo using mass cytometry. Plasma cytokines measured following treatment with ruxolitinib remained markedly abnormal, indicating that aberrant cytokine production persists despite therapeutic JAK2 inhibition. In MF patient samples, 14/15 cytokines measured by mass cytometry were found to be constitutively overproduced, with the principal cellular source for most cytokines being monocytes, implicating a non-cell-autonomous role for monocyte-derived cytokines impacting disease-propagating stem/progenitor cells in MF. The majority of cytokines elevated in MF exhibited ex vivo hypersensitivity to thrombopoietin (TPO), toll-like receptor (TLR) ligands, and/or tumor necrosis factor (TNF). A subset of this group (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TLR/TNF-sensitive cytokines, however, were sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling. These results indicate that NFκB and MAP kinase signaling maintain cytokine overproduction in MF, and that inhibition of these pathways may provide optimal control of inflammatory pathophysiology in MF.

Inflammatory cytokine production is characteristic of myeloproliferative neoplasms including myelofibrosis (MF). Clonal dominance and bone marrow fibrosis have been attributed in part to cytokine overproduction. Elevated plasma levels of several cytokines have been associated with decreased survival in MF patients; notably IL-8/CXCL8, which has been associated with transformation to secondary acute myeloid leukemia (sAML; Tefferi et al., 2011, J. Clin. Oncol.). In MF patients, JAK2 inhibition with ruxolitinib ameliorates constitutional symptoms and splenomegaly; but improvements in anemia, fibrosis, and malignant clonal burden are infrequent, and survival benefit is modest. We have measured blood plasma cytokines in MF patients prior to and on treatment with ruxolitinib, versus healthy controls and sAML patients. Cytokines significantly elevated in MF patients versus controls (VEGF, TNF, IL-6, IL-10, IL-16) showed minimal differences in pre-versus-on ruxolitinib levels. Indeed, both pre- and on ruxolitinib cytokine levels in MF were similar to those observed in sAML. Therefore, overall cytokine-producing activity in MF is of similar magnitude to that in leukemic hematopoiesis, and persists despite ruxolitinib treatment. To identify cell populations responsible for cytokine production, MF blood cells were studied using mass cytometry (CyTOF). In MF versus healthy control samples, 14/15 cytokines measured were found to be constitutively overproduced in myeloid cell populations. IL-8 was highly expressed in CD34+ hematopoietic stem and progenitor cells (HSPC), which are prevalent in MF blood. The prognostic impact of IL-8 may therefore reflect overproduction of phenotypic HSPC, which could indicate incipient transformation to sAML. For the majority of MF overproduced cytokines, however, the largest source population was monocytes. This indicates that cytokines produced by monocytes may impact disease-propagating HSPC in a non-cell-autonomous fashion. To evaluate mechanisms driving cytokine overproduction, MF patient and control samples were exposed to 4-hour incubations with TPO, TNF, and/or TLR ligands PAM3CSK4 and R848. These compounds activate either JAK/STAT or NFκB signaling. The majority of cytokines elevated in MF exhibited hypersensitivity to these agents in MF versus normal monocytes. TPO induced cytokines in monocytes via its receptor MPL, which was expressed in a subset of both normal and MF monocytes. Cytokine induction by TPO was observed in normal and MF monocytes isolated by flow sorting (CD14+CD34-CD61-), confirming that MPL and JAK2 can induce cytokine overproduction in monocytes cell-autonomously. TPO induced cytokine production in monocytes could be suppressed either by ruxolitinib or pevonedistat, a NEDD8-activating enzyme inhibitor that blocks NFκB activation. This implies that both JAK2 and NFκB signaling pathways must be intact for maximal cytokine induction downstream of MPL. In contrast to TPO induced cytokine production, basal constitutively elevated cytokine production in MF monocytes was not consistently reduced by ruxolitinib. Among the TPO/TNF/TLR inducible cytokines, a subset (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TNF/TLR-sensitive cytokines, however, were at least partially sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling, as observed from incubation with pevonedistat, trametinib (MEK inhibitor), VX-745 (p38 MAPK inhibitor), and/or JNKi8 (JNK inhibitor). In contrast, these inhibitors did not suppress basal production of non-TPO-inducible cytokines (e.g. TGF-β, IFNγ), which are also overproduced in MF. These results indicate that overproduction of one set of cytokines in MF is driven by a combination of JAK/STAT, NFκB, and MAP kinase signaling; while that of another set is not directly driven by any of these pathways. Cytokines whose production is refractory to ruxolitinib may underlie features of MF that resist ruxolitinib therapy. A combined inhibitory approach may provide therapeutic benefit beyond that observed with ruxolitinib. Based on this hypothesis, our group has initiated a Phase 1 clinical trial combining pevonedistat with ruxolitinib for MF treatment (NCT03386214). Further results will indicate whether inhibition of NFκB confers an additional benefit to that observed from ruxolitinib alone. Disclosures Zhou: Incyte: Speakers Bureau. Oh:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Takeda: Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding.

Introduction: Hepcidin, a peptide-hormone produced by hepatocytes, is a key regulator of iron homeostasis in mammals. Hepcidin levels are affected by several factors including inflammation, iron concentration, and erythropoietic signaling, and elevated hepcidin levels can lead to anemia. Inflammatory cytokines such as IL-6 induce hepcidin mRNA transcription via JAK/STAT signaling, whereas high serum iron levels and high erythropoietic drive via erythroferrone (ERFE) suppress hepcidin transcription via BMP/SMAD signaling. Anemia is a common problem in patients with myelofibrosis (MF). In a previous study of primary myelofibrosis (PMF) patients, serum hepcidin levels were found to be elevated compared to normal controls and were associated with increased RBC transfusion requirement and reduced survival (Pardanani et al. Am J Hematol 2013;88(4):312-316). Ruxolitinib is a JAK1/2 inhibitor approved for treatment of patients with MF. Treatment with ruxolitinib reduces spleen volume, constitutional symptoms, and inflammatory cytokines, but does not lead to anemia improvement in MF (Verstovsek et al. NEJM 2012;366:799-807). To further assess the relationship between hepcidin and anemia in MF, we measured hepcidin levels in 99 MF patients including 49 PMF and 50 secondary MF (sMF) patients, as well as 24 patients treated with ruxolitinib. We also evaluated the relationship between hepcidin and hemoglobin levels, inflammatory cytokines, and serum iron markers. Methods: Hepcidin levels (ng/mL) were measured using a commercially available ELISA assay from Intrinsic Lifesciences (Intrinsic Hepcidin IDxTM ELISA) in 99 MF patient plasma samples and 9 normal controls. Plasma cytokine levels (IFN, IL-10, IL-2, IL-2Rα, IL-6, IL-8, MIP-1α, MIP-1β, TNFα, VEGF, TGF-β1, TGF-β2, TGF-β3) were measured via multiplex cytokine analysis (Mesoscale Discovery). CRP, transferrin saturation, and ferritin were measured using standard laboratory methods at our institution. Statistical analyses were performed using GraphPad Prism software. Results: Hepcidin was significantly elevated in MF patients compared to normals (median 81.1 vs 8.5, p Conclusion: These findings indicate that abnormal hepcidin production is a hallmark of both PMF and sMF. Hepcidin expression correlated with IL-6 and IL-2Rα, but not with other inflammatory cytokines, nor with CRP. These observations suggest a complex relationship between inflammation and hepcidin in MF. Although a significant correlation between hepcidin and hemoglobin was not found, this may have been related to the relatively small sample size. In addition, RBC transfusion in a subset of patients may have impacted the lack of correlation between iron indices and hepcidin. Hepcidin was high in patients treated with ruxolitinib, suggesting that suppression of JAK-STAT signaling (such as downstream of IL-6) via ruxolitinib may not be sufficient to normalize hepcidin production. Alternatively, inhibition of hepcidin production via ACVR1/ALK2 has been proposed to be the mechanism behind anemia improvement observed with momelotinib therapy (Asshoff et al. Blood 2017;129(13): 1823 - 1830). Therefore, the role of hepcidin as a therapeutic target for treatment of anemia in patients with MF warrants further exploration. Disclosures Zhou: Incyte: Speakers Bureau. Oh:Gilead: Research Funding; Janssen: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; CTI Biopharma: Research Funding.