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.

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Small molecule inhibitors targeting JAK2 provide symptomatic benefits for myeloproliferative neoplasm (MPN) patients and are among first‐line therapeutic agents. However, despite all having potent capacity to suppress JAK–STAT signaling, they demonstrate distinct clinical profiles suggesting contributory effects in targeting other ancillary pathways. Here, we performed comprehensive profiling on four JAK2 inhibitors either FDA‐approved (ruxolitinib, fedratinib, and pacritinib) or undergoing phase 3 studies (momelotinib) to better outline mechanistic and therapeutic efficacy. Across JAK2‐mutant in vitro models, all four inhibitors demonstrated similar anti‐proliferative phenotypes, whereas pacritinib yielded greatest potency on suppressing colony formation in primary samples, while momelotinib exhibited unique erythroid colony formation sparing. All inhibitors reduced leukemic engraftment, disease burden, and extended survival across patient‐derived xenograft (PDX) models, with strongest effects elicited by pacritinib. Through RNA‐sequencing and gene set enrichment analyses, differential suppressive degrees of JAK–STAT and inflammatory response signatures were revealed, which we validated with signaling and cytokine suspension mass cytometry across primary samples. Lastly, we assessed the capacity of JAK2 inhibitors to modulate iron regulation, uncovering potent suppression of hepcidin and SMAD signaling by pacritinib. These comparative findings provide insight into the differential and beneficial effects of ancillary targeting beyond JAK2 and may help guide the use of specific inhibitors in personalized therapy.

Prior studies by our group and others have demonstrated that platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. However, a complete understanding of platelet alterations in MPNs remains lacking, and the mechanisms by which platelets contribute to MPN-related thrombosis, as well as other MPN disease features, are incompletely understood. In this study, utilizing multiomic approaches to interrogate platelet phenotypes in patient samples, in conjunction with relevant MPN animal models, we aim to investigate mechanisms of dysregulated platelet activity in MPNs, and explore how these findings may be leveraged to uncover novel therapeutic strategies. We initially studied platelet activation in peripheral blood from patients with essential thromobocythemia (ET) and compared to age-, sex-matched healthy controls. We found significantly increased P-selectin exposure in conjunction with increased platelet-leukocyte aggregates indicating activation of platelets from ET patients. To investigate the transcriptional signature of PBMCs and platelets at the single cell level, we performed single cell RNA-seq (scRNA-seq) in PBMCs from ET patients and healthy controls. Monocytes were increased in ET patients and displayed the highest inflammation index, implicating monocytes as the primary source of inflammation in ET. Aside from the quantitative increases of platelets in ET patients, we demonstrated strong enrichment of platelet activation, mTOR and oxidative phosphorylation (OXPHOS) genes in platelets from ET patients. Proteomic data confirmed the activation of OXPHOS and mTOR pathways in platelets from MPN patients. Metabolomics analysis revealed distinct metabolic phenotypes consisting of elevated tricarboxylic acid (TCA) cycle components, ATP generation and lower alpha-ketoglutarate (alpha-KG), in platelets from MPN patients, all consistent with enhanced OXPHOS and mTOR activities. Enhanced mitochondrial respiration at both baseline and after ex vivo stimulation with the platelet agonist thrombin receptor activator peptide (TRAP6) by extracellular flux analysis confirmed the bioenergetic alterations in platelets from MPN patients. alpha-KG is a key TCA cycle intermediate, which inhibits PI3K/AKT/mTOR pathway signaling via suppression of ATP synthase. alpha-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients. We further investigated the effects of alpha-KG on MPN platelet activation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F knock-in mice with alpha-KG significantly reduced platelet surface P-selectin and integrin gpIIb/IIIa activation. Additionally, alpha-KG inhibited the spreading and adhesion of platelets from Jak2 V617F knock-in mice to fibrinogen-coated surfaces. Platelet phosphoblots demonstrated significant downregulation of p-AKT and p-ERK after treatment with alpha-KG, suggesting the involvement of PI3K/AKT/mTOR and MAPK pathways in the inhibitory effects of alpha-KG on platelet activation. Thus, alpha-KG inhibited platelet hyperreactivities in both human and mouse MPN samples. To test the therapeutic impact of alpha-KG on MPN disease features, we treated Jak2 V617F knock-in mice with alpha-KG for 6 weeks. Oral alpha-KG supplementation decreased splenomegaly and reduced elevated platelets and hematocrit. Additionally, monocytes were significantly decreased as early as 2 weeks after alpha-KG treatment in Jak2 V617F knock-in mice. Consistently, RNA-seq of bone marrow samples from alpha-KG treated mice revealed inhibition of heme metabolism, OXPHOS and mTOR signaling pathways. In ex vivo studies with MPN patient CD34+ cells, alpha-KG treatment for 10 days led to a decrease in CD41+ CD61+ cells, suggesting decreased megakaryocyte commitment. We further observed that alpha-KG incubation significantly decreased the secretion of proinflammatory cytokines from sorted CD14+ human monocytes. Mass cytometry analysis of whole blood from MPN patients demonstrated inhibition of MAPK pathway signaling after alpha-KG treatment. Taken together, these results suggest that alpha-KG supplementation may exert therapeutic effects through both direct inhibition of MPN platelet activity and via quenching of monocyte hyper-inflammation. In summary, these studies reveal a previously unrecognized metabolic disorder in platelets from MPN patients and highlight a prominent role for alpha-KG and mTOR signaling in aberrant MPN platelet activity and monocyte-driven inflammation. These findings have potential relevance for novel therapeutic approaches for MPN patients.

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.