Polycythemia vera is characterized by erythrocytosis and increased risk of thrombosis. Sapablursen is an antisense oligonucleotide that suppresses transmembrane serine protease 6 (TMPRSS6), increases hepcidin production, and reduces iron availability for erythropoiesis. IMPRSSION; an ongoing phase 2a, randomized, open-label study includes patients meeting 2016 WHO criteria for polycythemia vera, requiring ≥3 phlebotomies within 6 months of screening and ≥1 phlebotomy within the last 12 weeks. Cohort A (120 or 80 mg) and Cohort B (40 mg) received sapablursen subcutaneously once every 4 weeks (Q4W). The primary efficacy endpoint was the reduction in phlebotomy frequency from baseline to weeks 17-37 in patients who received ≥1 dose. Forty-nine patients received ≥1 sapablursen dose (median age 61 years; 82% male). The baseline mean±SD weekly phlebotomy rate was 0.15±0.07 in Cohort A; 0.17±0.07 in Cohort B and was reduced by -0.10 (95%CI, -0.13, -0.07; n=32;p<0.0001) in Cohort A; -0.10 (95%CI, -0.14, -0.06; n=17;p=0.0001) in Cohort B. Sapablursen reduced the median (IQR) number of phlebotomies from 5.0 (3.0-6.0,Cohort A; 3.5-6.5,Cohort B) at baseline (26-weeks) to 0 (0-1.0) in the efficacy evaluation window (20-weeks) in Cohort A and 1.5 (0-2.5) in Cohort B. Most adverse events were mild or moderate; anemia and fatigue were most common. The mean change in MPN-SAF-TSS at week 37 was -6.21 points (95%CI: -10.56, -1.85, p=0.0052) in Cohort A; -2.71 points in Cohort B (95% CI: -8.33, 2.91, p=0.34). Sapablursen reduced phlebotomy need, improved symptoms, and was safe and well-tolerated in patients with polycythemia vera. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov: NCT05143957)

Calreticulin (CALR) frameshift mutations drive the majority of JAK2/MPL-wild-type cases of essential thrombocythemia and myelofibrosis, producing a shared novel C-terminus that activates the thrombopoietin receptor and leads to constitutive Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. CALR mutations transform the multifunctional endoplasmic reticulum (ER)-resident protein into an oncogenic driver that aberrantly traffics to the cell surface, activates the thrombopoietin receptor, MPL, and promotes MF megakaryocytic proliferation and hemopoietic stem cell fitness. Clinically, CALR-mutated MPNs affect younger patients, exhibit thrombocytosis and progressive anemia, and, in MF patients, are associated with a superior survival compared with JAK2- or MPL-mutated disease, yet responses to hydroxyurea and ruxolitinib remain inferior. The C-terminal motif shared between CALR mutations is presented extracellularly on major histocompatibility complex (MHC) Class I molecules and in complex with the thrombopoietin receptor enabling targeting with vaccination and antibody based therapeutic platforms, respectively. Mutant CALR peptide vaccines induce T cell responses but have failed to result in hematologic or molecular responses. Mutant CALR-specific monoclonal antibodies, such as Fc-silent antagonists, can block mutant-CALR-MPL signaling, suppress hematopoietic stem and progenitor cells (HSPC) proliferation and megakaryopoiesis, and achieve rapid, durable hematologic remissions with minimal toxicity in early-phase trials. Preclinical antibody-drug conjugates, bispecific T-cell engagers, and chimeric antigen receptor T cell (CAR-T) cells also show potent, selective mutant-cell killing. Here we review the basis and ongoing translational and clinical efforts in the development of CALR-targeted immunotherapies that offer a potential shift from symptom management to disease-modifying treatment in this molecularly defined MPN subset.

Menin inhibition, an approved therapy for KMT2A-rearranged and NPM1 mutant acute leukemia, is accompanied by decreased platelet counts in 15-20% of heavily pre-treated patients. While studying the mechanism underlying this effect, we discovered that menin inhibition reduced the numbers of megakaryocyte progenitors in human CD34+ cultures and in mice. Because megakaryocytes are key drivers of myeloproliferative neoplasms (MPNs), we investigated the extent to which menin inhibition ameliorates MPN phenotypes. We found that the menin inhibitor revumenib has potent anti-tumor activity, synergizes with ruxolitinib, and shows only subtle effects on healthy mice. Moreover, revumenib suppressed megakaryopoiesis of primary MPN patient specimens in vitro and in vivo. Importantly, genetic knockout of MEN1 and its target MEF2C phenocopied the action of revumenib, confirming an on-target effect of the drug. Together, we reveal menin as a dependency in proliferative megakaryocytes and support further evaluation of menin inhibition as a potential therapy for MPNs.

The peptide hepcidin is produced by the liver and serves as the central negative regulator of iron trafficking. Recently, drugs that affect the hepcidin pathway have been evaluated as potential treatment options for both controlling the degree of erythrocytosis in polycythemia vera (PV) patients as well as correcting anemia associated with myelofibrosis (MF). Under normal conditions, increased hepcidin levels limit iron absorption from the gastrointestinal tract and iron recycling from liver and splenic macrophages, thus decreasing plasma iron levels and restricting iron availability for erythropoiesis. In PV, however, unrestricted erythropoiesis occurs despite low systemic iron levels. Since hepcidin levels are relatively low in PV patients, hepcidin agonists (rusfertide, divesiran, sapablursen) are undergoing clinical development to control PV associated erythrocytosis, thereby reducing the need for therapeutic phlebotomies and myelosuppressive therapeutic options. By contrast, hepcidin levels are increased in MF patients leading to the trapping of iron in tissue macrophages which creates a picture which resembles the anemia of chronic inflammation. A number of strategies to lower hepcidin levels (the JAK2 inhibitors pacritinib and momelotinib, anti-hemojuvelin monoclonal antibody DISC-0974C) are currently undergoing clinical development to make systemic iron available for erythropoiesis and alleviate the degree of MF associated anemia. These new therapeutic options that modulate iron trafficking in PV and MF patients represent the application of greater knowledge of iron trafficking to create novel therapeutic options to treat patients with hematological malignancies.

Fibrotic remodeling of the bone marrow (BM) niche is a characteristic feature of myelofibrosis (MF) that contributes to disease progression. In MF, mesenchymal stromal cells (MSCs) produce excessive amounts of inflammatory cytokines and extracellular matrix, leading to BM fibrosis, impaired blood production, extramedullary hematopoiesis, and progressive BM failure. While the genetic events that initiate MF in hematopoietic cells are well defined, our understanding of the mechanisms responsible for BM fibrosis remains incomplete. Here, we show that transcription factor EBF1 is a key regulator of the fibrotic gene program in mouse and human MSCs. EBF1 is upregulated in pre-fibrotic MSCs, while mice with MSC-specific deletion of Ebf1 exhibit reduced BM fibrosis, decreased expansion of myeloid cells and splenomegaly when transplanted with hematopoietic progenitors harboring the MF driver mutation MPL W515L . Moreover, we identify ITGB8 as an EBF1-regulated gene with therapeutic potential. MF mice treated with ITGB8-neutralizing antibodies or with MSC-specific Itgb8 deletion show reduced disease burden, as indicated by decreased marrow fibrosis, significantly reduced frequencies of MPL mutant cells, and reduced inflammation in the BM. Our data indicate that targeting the EBF1-ITGB8 axis in the MF MSCs may have therapeutic benefits.

Prefibrotic primary myelofibrosis (prePMF) and essential thrombocythemia (ET) are distinct myeloproliferative neoplasms (MPNs) with overlapping clinical features, often leading to diagnostic uncertainty. We developed an artificial intelligence (AI) framework with human interpretability to distinguish prePMF from ET using digitized hematoxylin and eosin-stained bone marrow biopsy (BMB) slides. Trained on an initial cohort of MPN patients with thrombocytosis, the AI model achieved an AUROC of 0.89 and accuracy of 92.3%. To assess the image features guiding predictions, we generated synthetic images which potentially exaggerate disease-specific morphologies. In a blinded survey, hematopathologists reviewed both real and AI-generated images. While human experts frequently agreed with AI predictions on diagnosis with real images, diagnostic discordance reached up to 88% for AI-generated ET images despite being correctly predicted by AI. We further quantified marrow cellularity and adiposity in the real and generated images, which revealed a higher proportion of fat content in all ET images (42.0%) compared to prePMF (28.9%). These findings suggest that AI can utilize morphological cues distinct from current established diagnostic criteria, such as proportion of adiposity to distinguish types of MPNs. Thus, an AI-assisted diagnostic tool underscores the potential of AI to augment histopathologic evaluation and allow identification of more specific subpopulations of forms of MPNs.

Myelofibrosis (MF) is a chronic myeloproliferative neoplasm (MPN) characterized by splenomegaly, constitutional symptoms, marrow fibrosis, cytopenias and inflammation. Janus kinase 2 (JAK2) inhibitors, such as ruxolitinib, reduce splenomegaly and alleviate symptoms but have limited disease-modifying activity, and resistance frequently develops. Selinexor, an oral selective exportin (XPO1) inhibitor, restores nuclear retention of tumour suppressors and inhibitors of inflammatory signalling. Dual XPO1/JAK2 inhibition targets complementary downstream pathways, enhancing suppression of MF progenitor cell fitness. We evaluated selinexor plus ruxolitinib in MF models, including JAK2V617F or calreticulin (CALR) exon 9-mutant MPN cell lines and samples from patients with MF. The combination showed greater anti-proliferative activity than JAK2 inhibition alone and suppressed colony formation from MF Cluster of Differentiation 34 (CD34)+ cells. Selinexor remained active in a ruxolitinib-resistant MPN cell line, inducing G1 arrest and apoptosis. Multi-omic analyses demonstrated increased nuclear retention of p53 and disruption of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling, complementing and extending ruxolitinib-targeted pathways. The combination suppressed NF-κB transcriptional activity and reduced Tumor Necrosis Factor alpha (TNFα), Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1) secretion from MF patient peripheral blood mononuclear cells. These findings highlight that selinexor plus ruxolitinib affects both MF haematopoietic cell-intrinsic and microenvironment-related pathways, providing a novel disease-modifying strategy with the potential to improve clinical outcomes in patients with MF.

Cancer develops through the interactions between cancer stem cells and the components of tumor microenvironment (TME). To model in vivo cancer stem cell/TME interactions and elucidate their functional consequences, we focused on myelofibrosis (MF), a stem cell driven myeloproliferative neoplasm. We co-cultured MF hematopoietic stem and progenitor cells (HSPCs) with normal donor endothelial cells (ECs) and mesenchymal stromal cells (MSCs) to investigate the consequences of interactions between malignant MF HSPCs and non-malignant microenvironmental cells. This tri-cultivation system proved to be a simple and reproducible platform, which promoted malignant clone dominance and the persistence of MF HSPCs that recapitulate the MF phenotype upon transplantation into immunodeficient mice, including splenomegaly and marrow fibrosis. Transcriptional profiling revealed extensive reprogramming of not only the co-cultured MF HSPCs, but also MSCs and ECs. Although numerous disease-relevant pathways were upregulated, the pro-inflammatory response stood out as a key consequence of MF HSPC/TME interactions. We validated these findings through quantitation of pro-inflammatory transcript upregulation and cytokine production. This human multicellular model system has proven useful in demonstrating the multidirectional interactions of MF HSPCs with TME cells that are essential for sustaining fully functional MF stem cells.

Myeloproliferative neoplasm (MPN) patients are at risk of splanchnic vein thromboses (SVT). Prior studies have failed to demonstrate a benefit of hydroxyurea therapy in preventing recurrent thrombosis after SVT, but are limited by small sample sizes and inability to account for starting, stopping, or switching of cytoreductive therapies. To better assess the impact of cytoreductive therapy on thrombosis recurrence, we performed a retrospective cohort study of MPN-SVT patients from the international GASTRO-MPN Consortium, treating cytoreductive therapy as a time-dependent covariate. We identified 757 patients, of which 43% were treated with cytoreductive therapy. During a median follow-up of 7.4 years, 256 patients (34%) had thrombosis recurrence; 145 (19%) had SVT extension/recurrence/TIPS thrombosis, 138 (18%) an thrombosis recurrence outside the splanchnic bed (74% were venous). Additionally, 262 patients (35%) experienced clinically relevant bleeding and 170 (22%) died. After adjusting for age, sex, prior thrombosis, timing of MPN diagnosis relative to SVT, anticoagulation initiation within 30 days of SVT diagnosis, MPN type, JAK2 status, other cancers and cirrhosis, cytoreductive therapy was associated with a decreased risk of recurrent thrombosis (HR 0.67 95%CI 0.50-0.89, p=0.01). This was driven by a lower risk of SVT extension/recurrence (HR 0.62 95%CI 0.42-0.90, p=0.01) but not by extra-splanchnic thrombosis recurrence (HR 0.97 95%CI 0.72-1.30, p=0.82). In a subgroup of patients without erythrocytosis/thrombocytosis at initial SVT (n=275), cytoreductive therapy was associated with a HR 0.65 (95%CI 0.40-1.07, p=0.09) for recurrent thrombosis. These data support the use of cytoreductive therapy in MPN-SVT patients to prevent recurrent thrombosis, particularly SVT extension/recurrence.

Myelofibrosis (MF) is a chronic, progressive myeloproliferative neoplasm characterized by bone marrow fibrosis, ineffective blood cell production, and neoplastic extramedullary hematopoiesis (EMH) occurring primarily within the spleen. To explore the molecular mechanisms underlying splenic EMH, we performed single-cell transcriptional and chromatin profiling of cells from MF spleens that had been surgically removed. We demonstrate significant expansion of hematopoietic stem and progenitor cells, coupled with aberrant differentiation toward the erythroid and megakaryocytic lineages, associated with a significant enrichment of inflammatory pathways with enhanced NF-κB signaling and IFN responses, as well as dysregulation of the inferred function of differentiation-defining transcription factors. Finally, we report a significant remodeling of the immune microenvironment in MF spleens, characterized by emergence of dysfunctional T cell subsets and inflammatory memory B cells, suggesting the concomitant establishment of a pro-inflammatory and immune-tolerant tumor microenvironment within the spleen that influences hematopoietic cell differentiation and impairs tumor immune surveillance.

Idasanutlin is a small molecule inhibitor that restores p53 activity, triggering apoptosis. Two trials (phase 1/2) of idasanutlin therapy in polycythemia vera patients that were intolerant or refractory to hydroxyurea resulted in substantial clinical and molecular responses. Here the long term outcomes of these patients are reported.

Ataxia telangiectasia mutated and Rad3-related kinase (ATR) mediates cellular responses to replication stress and DNA damage. Camonsertib is a potent and selective ATR inhibitor with promising clinical activity (NCT04497116) in cancer patients, but severe anemia is a common side effect of treatment. Iron is essential for heme synthesis during erythropoiesis but increases erythroblast vulnerability to iron-mediated oxidative damage. Iron accumulation in ferritin and nuclear coactivator 4 (NCOA4) mediated ferritinophagy are essential for normal erythropoiesis while during oxidative stress, iron release from ferritin promotes ferroptosis, a form of cell death resulting from iron-mediated lipid peroxidation. We hypothesize that camonsertib-induced anemia is due to increased ferroptosis occurring during terminal erythropoiesis. We previously showed that camonsertib leads to bone marrow erythroblast depletion in mice with reversal upon drug withdrawal. Currently, using primary cultures of human erythroblasts, we demonstrate that camonsertib induces a dose-dependent decrease in erythroblast number and an increase in reactive oxygen species (ROS) in erythroblasts. We also demonstrate that in camonsertib-treated erythroblasts, ferritin decreases while NCOA4 increases, consistent with enhanced ferritinophagy. Furthermore, camonsertib-treated erythroblasts exhibit a dose-dependent increase in lipid peroxidation and cellular iron accumulation. Finally, the expression of glutathione peroxidase 4 (GPX4), a key regulator of ferroptosis, is decreased in camonsertib-treated erythroblasts. Camonsertib-induced increase in ROS, lipid peroxidation, and NCOA4 expression are ameliorated by ferrostatin-1 (a ferroptosis inhibitor) and N-acetylcysteine (an antioxidant agent). Taken together, our results demonstrate ferroptosis induction in camonsertib-treated erythroblasts and may represent the underlying cause of ATRi-induced anemia in patients.

The JAK2 V617F (JAK2 VF ) driver mutation is found in 95% of patients with polycythemia vera (PV), a progressive myeloproliferative neoplasm. Current treatments suppress excessive hematopoiesis but lack specificity for targeting JAK2 VF cells, are unable to deplete mutant stem/progenitor cells and ultimately result in drug resistance. We discovered that the FDA-approved antibiotic, linezolid (LZD), ameliorates the PV phenotype across multiple model systems. LZD suppressed cell proliferation and STAT5 signaling, altered the cell cycle, and increased apoptosis of JAK2 VF -harboring human erythroleukemia cells, but not in wild-type acute leukemia cells. Computational modelling indicated that LZD interacts specifically with mutant JAK2 VF but not with wild-type JAK2 protein. We further showed that, in JAK2 VF mice that faithfully recapitulate human PV, LZD mitigates disease burden by selectively targeting JAK2 VF stem cells thereby normalizing spleen size and blood counts. LZD also inhibited hematopoietic colony formation by patient-derived peripheral blood mononuclear cells, with the more primitive progenitors being preferred targets. Importantly, LZD selectively decreased JAK2 VF+ colony numbers, without impacting wild-type JAK2 colonies. In all, the data provide a firm foundation for evaluating LZD-like molecules as an effective therapy for PV and other myeloproliferative neoplasms. Key points:Linezolid acts as a JAK2 V617F IZselective inhibitor in PV mouse models and PV patient samples while sparing wildIZtype hematopoiesis. Linezolid acts directly on JAK2 V617F hematopoietic stem cells.

Patient listing of prior myelofibrosis treatment and time since discontinuation if known

Menin inhibition has emerged as an exciting therapeutic strategy for acute myeloid leukemia associated with KMT2A-rearrangements and NPM1 mutations. While the FDA approved menin inhibitor revumenib is highly active and well tolerated, one potential side effect is thrombocytopenia. To study the mechanism of thrombocytopenia and determine whether this is an on-target effect of revumenib, we first cultured human CD34+ cells supplemented with thrombopoietin and treated with vehicle or revumenib under conditions to expand the megakaryocyte lineage. We observed a significant 5.7-fold decrease in the numbers of CD41+ megakaryocyte progenitor cells (MkP) as well as a decrease in the generation of mature, polyploid megakaryocytes with revumenib treatment. Single-cell RNA sequencing of these cultures demonstrated that revumenib selectively depleted megakaryocytes and megakaryocyte progenitor cells, while sparing other hematopoietic lineages, including megakaryocyte-erythroid progenitors, erythroid progenitors, common myeloid progenitors, and granulocyte-monocyte progenitors. To confirm on-target activity of revumenib, CRISPR-Cas9–mediated knockout of MEN1 in human CD34⁺ cells recapitulated the selective depletion of megakaryocytes, including a 2-fold decrease in MkPs. Mechanistically, we discovered that expression of the KMT2A-menin target gene MEIS1 is inhibited by revumenib, and that knockout of MEIS1 in human CD34+ cultures largely phenocopied the effects of revumenib. Our further observation that overexpression of MEIS1 could not rescue the effects of revumenib suggests that other target genes contribute to megakaryopoiesis. Of note, ziftomenib, a structurally distinct menin inhibitor currently in clinical trials, similarly affected megakaryopoiesis in vitro, indicating this is a class effect. Together, these findings provide strong genetic and pharmacologic evidence that menin plays a key role in megakaryopoiesis. Next, given that megakaryocyte hyperproliferation is a hallmark of the myeloproliferative neoplasms (MPNs), we hypothesized that menin inhibition might be therapeutically leveraged in this disease. We evaluated the therapeutic potential of revumenib in primary human MPN samples with CALR and JAK2 mutations. In vitro treatment of these MPN specimens resulted in a significant reduction of CFU-Mk and selectively suppressed the expansion of megakaryocytes and megakaryocyte progenitors in culture with a 2.7-fold decrease in MkPs. To assay the activity of revumenib in MPN in vivo, we studied the effect of the drug on disease in the MPLW515L, MPLS504N, and JAK2V617F mouse models. Mice were treated with revumenib-formulated chow, administered either as a single agent or in combination with ruxolitinib. In the MPLW515L and MPLS504N models, revumenib monotherapy led to a significant reduction in leukocyte and platelet counts, a 5.7-fold decrease in megakaryocyte numbers in the bone marrow, and ablation of bone marrow fibrosis. In the JAK2V617F model, revumenib normalized hemoglobin and hematocrit, reduced spleen weight by 4.3-fold, and eliminated bone marrow fibrosis. Of note, combined treatment with revumenib and ruxolitinib further enhanced the suppression of leukocytosis, thrombocytosis, and erythrocytosis, normalized the spleen weight with a 5.8-fold decrease, and suppressed development of bone marrow fibrosis across these models. The reduction in fibrosis was accompanied by a robust downregulation of fibrogenic cytokines, most notably a 6.3-fold decrease in TGF-β. Importantly, revumenib as a single agent and in combination with ruxolitinib also resulted in marked, significant improvement in overall survival. Finally, revumenib was well tolerated in a 12-week study of healthy mice. Together these findings demonstrate that revumenib suppresses megakaryopoiesis both in vitro and in vivo in MPN models and highlight menin inhibition as a potential disease-modifying strategy capable of reversing key pathological features of MPNs.

Background: Janus Kinase (JAK) inhibitors are the current standard of care for patients (pts) with myelofibrosis (MF). However, many pts may not achieve spleen volume reduction (SVR) or total symptom score (TSS) response after frontline treatment, and most pts with relapsed/refractory (R/R) MF lack adequate responses. Momelotinib (MMB), a recently approved JAK/ACVR1 inhibitor for MF pts with anemia, showed symptom and spleen responses in about 25% pts in R/R setting. Combination strategies of JAK inhibitor and agent with unique mechanism of action and minimal overlapping toxicities (e.g. cytopenias) are needed to improve response rates in MF. PIM1 expression is upregulated in MF CD34 cells. In preclinical models, PIM1 knockout (KO) was shown to prevent MF progression without affecting PLT counts, whereas pan-PIM KO caused thrombocytopenia (TCP). Nuvisertib (NUVI, TP-3654), an oral investigational highly selective PIM1 kinase inhibitor, alone and in combination with ruxolitinib (RUX) showed spleen size reduction and bone marrow (BM) fibrosis improvement in JAK2V617F and MPLW515L MF mouse models. Preliminary data from the ongoing Phase 1/2 study in R/R MF pts with PLT count ≥25 x 109/L showed that NUVI monotherapy was well tolerated with limited myelosuppression, and clinical activity including SVR and TSS responses strongly correlating with cytokines modulation, and hemoglobin (Hgb), PLT, and BM improvement. Preclinical and monotherapy clinical data support the development of NUVI + MMB combo in MF. Methods: The global Phase 1/2 study evaluates the safety and efficacy of NUVI + MMB combo in pts with MF (NCT04176198, Arm 3). Key eligibility criteria include primary or secondary MF, previously treated with JAK inhibitor, DIPSS intermediate or high-risk MF, Hgb <10 g/dL, PLT ≥50 x 109/L, splenomegaly (≥450 cm3 by imaging), and ≥2 measurable symptoms with each score ≥3 or a total average score of ≥10 per MFSAF v4. The study aims to identify the RP2D of NUVI when given with MMB, and to assess the safety, clinical activity (SVR, TSS improvement), and PK and PD markers (cytokine, BM fibrosis etc.). Results: Here we present the first ever combination data of MMB in MF. As of 29 May 2025, total 18 pts enrolled in 4 dose levels of NUVI BID at 240 mg (n=4), 360 mg (n=8), 480 mg (n=5) and 720 mg (N=1) + MMB 200 mg QD using the BLRM dose escalation. At baseline, median age 75 years (range 51, 82); TSS 29 (9, 37); spleen volume 1370 cm3 (614, 4250); Hgb 9.1 g/dL (7.9, 10.1; 50% pts required transfusion); and PLT 196 x 109/L (81, 601). All pts received prior JAK inhibitor, and 53% pts had high molecular risk mutation. Median treatment duration of NUVI + MMB combo was 21 weeks (1, 30), and 13 of 18 (72%) pts were on treatment. One DLT of Grade 4 TCP without any bleeding occurred in NUVI 360 mg BID + MMB 200 mg QD dose. Treatment-related adverse events (TRAEs) occurring in ≥20% of pts were diarrhea, nausea, and TCP. Grade ≥3 TRAE occurring in ≥2 pts included TCP (n=2; 1 pt had baseline TCP). Mean Hgb and PLT remained stable throughout the 24-week treatment. Emerging NUVI + MMB combo safety data was generally consistent with NUVI monotherapy data. 5 pts in the NUVI 360 mg BID + MMB 200 mg QD dose completed ≥24 weeks of treatment and were considered efficacy evaluable. TSS improvement at WK24 was observed in all 5 patients (median change -65%, range -38% to -72%); 3 of 5 (60%) pts showed ≥50% TSS reduction. In addition, absolute reduction was observed in all 7 symptom parameters, including >50% reduction in mean fatigue score at WK24. 2 of 5 (40%) pts showed ≥25% SVR at WK24. Decreased EN-RAGE and increased adiponectin were observed in all 5 pts, consistent with NUVI monotherapy findings where modulation of these cytokines strongly correlated with TSS50, individual symptoms and SVR25 responses. Anemia improvement was observed in 2 of 5 (40%) pts during 24 weeks of treatment: 1 pt showed Hgb response (defined as mean ≥1.0 g/dL increase for ≥12 weeks without transfusion), and 1 pt achieved a >50% reduction in transfusions. Dose escalation is ongoing, and updated data will be presented.Conclusions: NUVI + MMB combo appeared to be well tolerated. Preliminary data showed early clinical activity including 60% TSS50 response and absolute symptom improvement, 40% SVR25 response, cytokine modulation and anemia improvement in R/R MF pts with anemia. Preliminary data supports further development of NUVI + MMB combo for pts with MF.

Background: The functional importance of bone marrow adipocytes (BMA) is frequently overlooked even though they are critical components of the BM (bone marrow) microenvironment. BMA have been proposed to play a supportive role within the hematopoietic niche and to directly sustain hematopoietic stem cell (HSC) survival as well as to preferentially support leukemia cell survival and proliferation (Sabbah Cell Commun Signal 2023). Although significant ex-vivo data implicates direct and indirect crosstalk between BMA and neoplastic HSCs (Venti Front Endocrinol 2022), the possible role of BMA in myeloproliferative neoplasms and their dynamics following therapy have yet to be characterized. Adiponectin, an anti- inflammatory adipokine that maintains metabolic homeostasis through regulation of insulin sensitivity, lipid metabolism and pro-inflammatory signaling is low in MPN patients and increases with IFN-α therapy (Avcu Int J Hematol. 2006). IFN-α has also been implicated in inhibiting adipogenesis via JAK-STAT1 signaling (Lee Biochim Biophys Act. 2016). A single institutional study of IFN-α in patients with essential thrombocythemia (ET) and polycythemia vera (PV) confirmed the potential for BM histopathologic responses and a correlation with molecular responses (Masarova Exp Hematol Oncol 2017). The aim of this analysis was to characterize the change in BM cellularity and fat composition with pegylated IFN α-2a (Pegasys; PEG) and the relationship with both hematologic and molecular response from tissue collected in prospective, global clinical trials of PEG in patients with ET/PV. Methods: A total of 24 subjects from the phase 2 MPN-RC 111 (Yacoub Blood 2019) salvage PEG study and 16 subjects from the MPN-RC 112 (Mascarenhas Blood 2022) phase 3 study comparing PEG to HU with available clinical, BM H&E slides, laboratory and molecular data were included in this retrospective analysis of prospective clinical trial data. We assigned a semi-quantitative score for changes in cellularity, fat distribution, and megakaryocyte (MK) histo-topography for changes from baseline, at 12, 24 and 36 months when available. Changes in BMA content and para-trabecular distribution were assessed on whole slide digital images with machine learning models. BMA content was quantified with a QuPath pixel classifier that distinguished adipocytes from other marrow components (Bankhead Scientific Reports 2017). Para-trabecular adipocyte distribution was assessed by leveraging UNI2-h, a self-supervised Vision Transformer (ViT-H/14) model and successor to UNI (Mahmood Nature 2024). UNI2-h was pre-trained on 200 million image tiles from over 300,000 H&E and IHC slides. In this study, UNI2-h was adapted to distinguish between high and low para-trabecular adiposity using image tiles centered on trabeculae. Trabeculae with a surrounding layer of adipocytes were labeled as ‘high’ adiposity, while ‘low’ para-trabecular fat distribution was defined by the direct contact of cellular marrow with trabeculae, lacking surrounding adipocytes. Results: A total of 40 PEG-treated ET (n=18) and PV (n=22) patients were included in this analysis with a median age of 64 years (26-85), 77% JAK2V617F+, 23% CALRmut+, and 40% treatment-naïve. The median JAK2V617F variant allele fraction (VAF) was 26% (range 3-96%). A complete hematologic response (CHR) was obtained with PEG in 58%, 61% attained >20% reduction in driver VAF and 48% attained a >50% reduction in VAF (MR) within 24 months of baseline. We observed an increase in BMA content and notable increase in para-trabecular distribution (confirmed by morphology and ML, independently) in 93% and 53% of PEG treated patients attaining a MR versus only 31% and 6% who did not attain MR, respectively (P Value=0.0004). There was more notable improvement towards normalization of MK histo-topography and orderly erythropoiesis in MR (86.6%) than in non-MR (31.2%) (P value=0.001). Increase in BMA content, BMA para-trabecular distribution, or improvement in MK histo-topography were not associated with achievement of CHR or changes in CALR VAF in this small cohort. Cytokine correlation with BMA dynamics is currently ongoing and will be reported at the meeting. Conclusion: Treatment of MPN patients with IFN-α resulted in reproducible changes in BM cellularity including MK histo-topography, erythropoiesis, BMA number and distribution. Changes in BMA were closely associated with reduction in JAK2V617F VAF following PEG treatment.

Introduction Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) for which the only approved therapies are JAK inhibitors which improve splenomegaly and disease-related symptoms. Inflammatory pathways beyond JAK-STAT are critical in the pathobiology of MF and contribute to patients' clinical manifestations. IL-1β is a master regulator of inflammation which is overexpressed in MPNs, preferentially supports the MPN mutant stem cell, and interacts with key cytokines that are implicated in fibrogenesis, inflammatory symptoms, and disease progression. Knockout of IL-1β in MPN mouse models led to an abrogated clinical phenotype, while pharmacologic inhibition of IL-1β resulted in reduction of reticulin fibrosis and osteosclerosis. Canakinumab is a fully human monoclonal antibody that binds to and neutralizes IL-1β. It is approved for rare inflammatory disorders with a safety profile that has been extensively studied in the CANTOS trial that assessed cardiovascular outcomes in high-risk patients (pts). Based upon these data, we designed a phase 2 study of canakinumab in pts with MF. Methods NCT05467800 is a Myeloproliferative Neoplasm Research Consortium (MPN-RC) ongoing open-label, 2-part, phase 2 study of canakinumab in pts with MF. Part 1 enrolled pts with MF previously treated with or ineligible to receive a JAK inhibitor. Canakinumab was given subcutaneously at a dose of 200mg on day 1 of 21-day cycles. Primary response assessment occurred at week 24 following cycle 8. Responding pts could continue to receive canakinumab. Eligible pts had platelets >25 x109/L and a need for MF-directed therapy defined as ≥ 1 of the following: hemoglobin <10 g/dL, transfusion dependency, splenomegaly palpated ≥ 5cm below left costal margin, and/or MPN-SAF version 4.0 TSS ≥ 10. The primary endpoint was overall response rate (ORR) including CR, PR, or CI after 8 cycles (24 weeks). A Bayesian optimal phase 2 (BOP2) design was used with an interim analysis performed after 10 pts. Results In part 1, 14 pts were enrolled and 13 were treated. One pt discontinued prior to starting treatment due to rapidly progressive disease. Enrollment onto part 1 has closed with 2 pts remaining on treatment. Median age was 70 y (range: 55-83). Eleven (84.6%) pts had received prior ruxolitinib. Median hemoglobin was 8.2 g/dL (range: 5.7-13.2), median platelet count was 114 x 109/L (range: 21-484), and median WBC was 9.7 x 109/L (range: 2.4-71.4). Five (38.5%) pts were transfusion dependent. Median MF-SAF TSS at baseline was 15 (range: 4-46). Median spleen volume at baseline was 1522 cm3 (range: 362cm3-2997cm3). Pts received a median of 9 cycles (range: 2-19). Three (23%) pts responded at week 24, achieving a CI-symptom response (TSS50). Six (46%) pts experienced progression of disease due to spleen volume increase. Median hemoglobin was 8.2 g/dL at baseline, 7.9 g/dL at week 12, and 9.0 g/dL at week 24. Median platelet count was 114 x 109/L at baseline, 98 x 109/L at week 12, and 183 x 109/L at week 24. Median MF-SAF TSS declined from 15 at baseline to 10 at week 12 and 9 at week 24. Eleven (85%) pts exhibited symptom improvement during treatment (MF-SAF TSS percent change from baseline range -20% to -85%). In pts with baseline hsCRP ≥ 1.0 (n = 8), canakinumab led to a reduction at week 24 (or EOT if prior to week 24) in 5/8 (62.5%) with a median decrease in CRP of 37%. The most common adverse events (AEs) were anemia and thrombocytopenia and often attributed to underlying disease. Non-hematologic grade ≥ 3 AEs occurred in 3 pts and were deemed unrelated to canakinumab, except for one grade 3 increase in bilirubin that was possibly related per the treating physician. Conclusion The IL-1β inhibitor, canakinumab, resulted in consistent symptom improvement and exhibited beneficial impact on cytopenias in pts with R/R MF while demonstrating a manageable toxicity profile. Consistent with pre-clinical data, inhibition of IL-1β had little impact on splenomegaly, an observation that was likely enhanced by frequent JAK inhibitor discontinuation prior to study start. Given the encouraging impact on MF symptoms, blood counts and reassuring safety profile, the study was amended to enroll pts on a stable dose of a JAK inhibitor (ruxolitinib, fedratinib, momelotinib, or pacritinib) who have the potential to benefit from additional therapy. Part 2 is currently enrolling at multiple sites and updated clinical and correlative data will be presented at the meeting.