Myelofibrosis Patients Research Articles

Real-world outcomes with fedratinib therapy in patients who discontinued ruxolitinib for primary myelofibrosis.

Aim: Fedratinib is an oral selective JAK2 inhibitor approved in the USA for the treatment of adult patients with intermediate-2 or high-risk primary or secondary myelofibrosis (MF). Methods: This observational study assessed adult US patients who received ruxolitinib for primary MF (Flatiron Health database: 1January 2011-31 October 2020). Patients were stratified by post-ruxolitinib treatment (fedratinib vs non-fedratinib). Results: Characteristics were comparable between fedratinib (n=70) and non-fedratinib (n=159)groups (median age: 71.0 vs 70.0years; females: 55.7 vs 50.3%; median follow-up: 7.0 vs 6.0months). Median overall survival (not reached vs 17months) and 12month survival (71.6 vs 53.5%) were improved with fedratinib versus the non-fedratinib therapies. Conclusion: In MF patients who received frontline ruxolitinib, survival was improved with subsequent fedratinib versus non-fedratinib care.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Positive-DAT and autoimmune manifestations in patients with JAK2V617F mutation

Objective: Myeloproliferative neoplasms (MPN) are occasionally associated with autoimmune manifestations. The prevalence of positive direct antiglobulin test (DAT) in patients with JAK2V617F mutation is not yet known. Methods: We conducted a cohort study of all consecutive patients with JAK2V617F mutation who underwent DAT at presentation from 2010 to 2022. We also looked for the prevalence of autoimmune diseases (AIDs) and relation to thrombosis. Results: Fifty nine patients formed the cohort, 22 females and 37 males, with a mean age of 65 years. The median follow-up time was 90 months. A positive-DAT was found in 6 to 7% of essential thrombocythemia (ET) and polycythemia vera (PV) patients, 60% of primary myelofibrosis (PMF) patients and 50% of myelodysplasia/myeloproliferation (MDS/MPN) patients with or without anemia. Autoimmune manifestations were present in 13% of patients with PV and 50% of those with MDS/MPN features. Overall, 25% of patients had autoimmune phenomena including the 15% with positive-DAT. Conclusion: In patients with JAK2V617F mutation, those with PMF and MDS/MPN subtypes are most likely to display a positive-DAT results and autoimmune manifestations (≥ 50%). Patients with PV and ET are less likely to exhibit autoimmune manifestations (≤ 10%). No correlation with the occurrence of thrombosis was seen.

Atrial Fibrillation in Patients with Myelofibrosis

Background: Atrial fibrillation (AF) is the most common arrythmia (1-2% of the general population) and is associated with high risk of arterial thrombotic events. AF is not well characterized in the context of myeloproliferative neoplasms (MPN). Recent studies imply that thrombotic risk stratification among MPN AF patients may be suboptimal using the standard CHA2DS2VASC score, and true predictor of thrombotic risk may be presence of myelofibrosis instead. Clinical context in which AF occurs in myelofibrosis patients had not been investigated so far, but such analysis is needed to better understand why is the presence of myelofibrosis in particular the main predictor of thrombotic events among AF MPN patients. We aimed to evaluate prevalence and clinical associations of AF among patients with myelofibrosis. Methods: We retrospectively investigated a cohort of 154 patients with overt myelofibrosis treated in 6 Croatian hematology centers in period 2004-2022. Diagnoses were reassessed according to the 2016 WHO criteria for primary myelofibrosis (PMF) and the 2008 IWGMRT criteria for secondary myelofibrosis (SMF). Presence of AF documented in medical records and clinical correlations of AF status with main disease related characteristics were analyzed. Results: Among a total of 154 patients, 86 had PMF, 35 had post polycythemia vera (PV) and 33 had post essential thrombocythemia (ET) SMF. Median age was 69 years. There were 91 (59%) males, 111 (75%) patients were JAK2, 9 (7.3%) CALR and 4 (3.3%) MPL mutated. AF was present in 15 (9.7%) patients with myelofibrosis which was significantly more frequent than anticipated in the general population of similar age (P<0.001). There were no significant associations of AF with neither etiology of myelofibrosis, age, sex, mutational status, degree of bone marrow fibrosis, spleen size, history of thrombosis, estimated glomerular filtration rate, hemoglobin, WBC nor platelet count (P>0.05 for all analyses). DIPSS score did not differ between AF and non-AF PMF patients, but MYSEC-PM score was significantly higher among AF SMF patients (P=0.029). AF in comparison to non-AF patients more frequently had constitutional symptoms (87% vs 54%, P=0.015), positive bleeding history (27% vs 6%, P=0.004), heart failure (47% vs 12%, P<0.001), obesity (21% vs 4%, P=0.010), lower MCHC (313 vs 318 g/L, P=0.031), higher urea (median 8.5 vs 6.8 g/L, P=0.009) and higher CHA2DS2VASC score (4 vs 3 points, P=0.003). AF more frequently received anticoagulants and diuretics than non-AF patients (P<0.05), whereas similar rates of cytoreductive therapy and aspirin were utilized. Despite all myelofibrosis patients with AF patients having CHA2DS2VASC score ≥2 points, only 60% of them received anticoagulant therapy, 27% received aspirin and 73% received cytoreductive therapy. No significant difference in survival and thrombosis was observed between AF and non-AF myelofibrosis patients (P>0.05 for all analyses) although study may be underpowered for these analyses. Summary/Conclusion: AF is frequent in patients with myelofibrosis and might be associated with higher frequency of constitutional symptoms, particular cardiovascular comorbidities and consistently high predetermined CHAD2DS2VASC risk. Nevertheless, unexpectedly low proportion of patients receives anticoagulant therapy, possibly due to anticipated risk of bleeding and cytopenias common in myelofibrosis patients. It remains a question whether suboptimal treatment or presence myelofibrosis itself might be a risk factor for unfavorable outcomes among AF MPN patients.

Thrombocytopenia Associated with Myelofibrosis Is Characterized By a Reduced Proportion of Megakaryocyte (MK) Biased Hematopoietic Stem Cells, Defects in MK Maturation and Increased Levels of Pro-Inflammatory Cytokines

The clinical course of patients with advanced forms of myelofibrosis (MF) can frequently be punctuated by thrombocytopenia. Marrow biopsies from these MF patients uniformly reveal megakaryocytic (MK) hyperplasia and dysplasia irrespective of the patient's platelet count. We have performed studies to better understand the origins of MF associated thrombocytopenia. We first examined the expression of G6b-B (MK-lineage specific immunoreceptor tyrosine-based inhibition motif) which is essential in promoting MK maturation, normal platelet release, platelet size and function (Becker. Blood Adv 2022). Recent reports have indicated that genetic deletion in G6b-B and loss-of-function mutations in both murine models and man lead to severe macro-thrombocytopenia and increased bone marrow fibrosis (Becker. Blood Adv 2022; Hofmann. Blood 2018; Melhem. Eur J Hematol 2017), indicating a possible role for this receptor in the development of thrombocytopenia in MF patients. We generated populations of MKs from mononuclear cells or CD34 + from thrombocytopenic or non-thrombocytopenic MFs and healthy donors (HDs) using a two-step culture system as previously described (Mosoyan. Leukemia 2017). A progressive increase of MK maturation (CD41 +CD42 +) was observed in HDs (28.5%±7) and non-thrombocytopenic MFs (32%±13.6) with a concomitant increase of total G6B expression 27%±9 and 38%±14 respectively. By contrast, MKs generated from thrombocytopenic MFs patient cells had limited CD41 +CD42 + (7%±5) and G6B (8.7%±4.7) expression (p<0.05). In addition, morphological analysis revealed that MKs from thrombocytopenic MF patients were small with hypolobulated nuclei while the MKs generated from HDs and non-thrombocytopenic MFs were larger and contained greater numbers of lobes ( Figure 1A). Psaila and coworkers have previously reported that MF is characterized by MK biased hematopoietic stem cells (HSCs) identified by increased expression of G6B (Psaila. Mol Cell 2020). We, however, determined that a reduced proportion of CD34 +CD38 - HSCs from thrombocytopenic patients expressed G6B as compared to non-thrombocytopenic MF or HD CD34 +CD38 - cells (p<0.05) ( Figure 1B). These studies indicate a reduced proportion of MK biased HSCs in thrombocytopenic MFs which likely leads to impaired MKs maturation. We next explored if the MF associated pro-inflammatory milieu also contributes to the MF MK maturation block. Thrombocytopenic MF plasma contained higher levels of YKL-40 (chitinase-3-like-1 protein), TNFα and TPO than HD plasma. For instance, MF plasma was characterized by higher levels of YKL-40 (median141,321 pg/mL) in comparison to HDs (median 19,380 pg/mL). Furthermore, YKL-40 levels were significantly higher in thrombocytopenic (median 187,696 pg/mL; p<0.05) than non-thrombocytopenic (median 78,082 pg/mL) MFs. In addition, TNFα and TPO plasma levels were also significantly higher in plasma from thrombocytopenic than non-thrombocytopenic MFs (p<0.05). We also observed that addition of either rYKL40 or rTNFα to HD-MK cultures resulted in delayed MK maturation as assessed by a reduced proportion of cells expressing CD41 +CD42 + (from 30% to 16.5% and 6.5% respectively) and G6B (from 35.5% to 22.5% and 9.5% respectively). We have previously reported that the treatment of MFs with a protein trap for TGFβ1 led to increases in platelet numbers (Mascarenhas. Clin Cancer Res 2023). The plasma from these patients prior to treatment not only had elevated levels of TGFβ1 (median 6433,917 pg/mL) but also YKL40 (median 275,640 pg/mL). Treatment of the patients with the TGFβ protein trap reduced both TGFβ1 (median 608,087 pg/mL) and YKL-40 (median 160,733 pg/mL) plasma levels, suggesting a regulatory loop between YKL-40 and TGFβ. We further explored this loop by treating the JAK2V617F+ HEL cell line with rYKL-40 in combination with rTGFβ1, which resulted in greater expression of YKL-40 (1.6 FI) than treated cells with rYKL-40 alone. In addition, treatment of HEL cells with a pan TGFβ inhibitor reduced YKL-40 protein expression (1.55 to 0.38). These data suggest the existence of an interplay between YKL40 and TGFβ on MF-MK maturation. Overall, these experiments indicate that thrombocytopenia in MF patients is due to intrinsic abnormalities in G6B originating at the level of the HSC compartment resulting in impaired MK maturation which can be further enhanced by the actions of a number of pro-inflammatory cytokines.

HMR Mutations Drive Poor Prognosis in Myelofibrosis Patients with Lower JAK2V617F Allele Burden but Not in Those with Higher Allele Burden: Results of a Multicenter Study

JAK2V617F is the most common driver mutation in myelofibrosis (MF), followed by CALR and MPL alterations. Clinical variables included in the International Prognostic Scoring System (IPSS), Dynamic IPSS (DIPSS), and DIPSS plus, as well as JAK2V617F allele burden are all well-established prognostic factors influencing the survival of MF patients. In addition, ASXL1, EZH2, SRSF2, IDH and U2AF1 mutations are classified as high-molecular risk (HMR) mutations in this context because of their close association with disease progression and leukemic transformation. However, the relationship between JAK2V617F allele burden and HMR mutations remains unclear. To address this, we recruited 122 MF patients (discovery cohort) diagnosed at the National Taiwan University Hospital (Taipei, Taiwan) who had available mutation profile determined by next-generation sequencing. For external validation, 82 MF patients from the Christie Hospital (Manchester, U.K.) and 51 MF patients from the Cleveland Clinic (Ohio, U.S.A.) and Vilnius University Hospital Santaros Klinikos (Vilnius, Lithuania) were enrolled. The median age of the discovery cohort was 61 years. JAK2V617F was the most common driver mutation (64.8%), followed by CALR (18%) and MPL mutations (9%). ASXL1 mutation was the most common (37%) genetic alteration other than driver mutations, followed by TET2 (16%) and EZH2 mutations (12%). Overall, 55 patients (45%) had at least one HMR mutation ( ASXL1, EZH2, SRSF2, IDH and U2AF1). With a median follow-up of 28.2 months, the median overall survival (OS) of the discovery cohort was not reached (NR) and was well risk-stratified by current prognostication systems, including IPSS, DIPSS plus, MIPSS70, and GIPSS. Notably, patients with at least one HMR mutation had significantly shorter leukemia-free survival (LFS) and OS than those without (both p<0.001). Next, we divided the 79 JAK2-mutated patients into lower- and higher-allele burden groups ( JAK2low and JAK2high) using the median (75%), as the cutoff. Concurring with previous reports ( Tefferi et al, Leukemia 2008; and Guglielmelli et al, Blood 2009), JAK2low patients had significantly inferior OS than JAK2high patients (median, 53 months vs NR, p=0.037). Among all JAK2-mutated patients, those with HMR mutations had a significantly shorter OS than those without (50 months vs NR, p<0.001). When considering HMR mutations and JAK2V617F allele burden together, the presence of HMR alterations conferred worse LFS and OS in JAK2low but not in JAK2high patients (HMR vs without HMR among JAK2low patients, p<0.001 and p<0.001; and JAK2high, p=0.822 and p=0.699, respectively). By and large, patients with concurrent HMR mutations and lower JAK2V617F allele burden had a distinctively inferior OS than those with other constellations of genomic lesions ( p<0.001, Figure 1A). This group of patients also had the shortest survival among the total cohort including both JAK2-mutated and wild patients ( p<0.001). The discriminating power of integrating HMR/ JAK2 allele burden for LFS and OS remained valid in the UK cohort (HMR vs without HMR: JAK2low, p=0.013 and p=0.012; and JAK2high, p=0.337 and p=0.600, respectively) and the US/Lithuania cohort (HMR vs without HMR: JAK2low, p=0.016 and p=0.013; and JAK2high, p=0.643 and p=0.446, respectively). Co-occurrence tests in the discovery and validation cohorts revealed that there was no shared mutational co-occurrence pattern among the 3 cohorts, excluding the potential confounding influence of specific concurrent mutations. More strikingly, time-dependent ROC curve analyses suggested that integrating JAK2 allele burden with HMR mutations could be complementary to current risk stratification systems in predicting LFS and OS of MF patients (Figure 1B) across 3 cohorts. Finally, HMR/ JAK2low was identified as an independent adverse prognostic factor by multivariable analysis, irrespective of age, risk stratification systems, and other mutations. In summary, we observe that HMR mutations accompanied by a lower mutant JAK2V617F allele burden identified a distinct MF population with significantly reduced survival and high risk of leukemic transformation, with independent prognostic relevance extrapolated to and validated in two independent cohorts. Experimental studies are ongoing to ascertain the underlying mechanistic basis and resolve the clonal architecture of this distinct MF genomic grouping.

Retrospective Analysis of the Relationship between Transfusion Independence and Bone Marrow Fibrosis Reduction in Patients with Myelofibrosis Treated with Pacritinib Versus Ruxolitinib

Background Pacritinib is a JAK1-sparing JAK2/IRAK1/ACVR1 inhibitor for treatment of myelofibrosis (MF). In addition to improving spleen volume and symptoms, pacritinib is associated with anemia benefit in MF patients. Recent in vivo studies have shown that dual JAK2/IRAK1 inhibition is associated with improvement in both cytopenias and bone marrow reticulin fibrosis (BMF) in an inflammation-driven murine MF model ( Cuenca Zamora E, et al. EHA 2023; P987 and P990). Here, we retrospectively analyzed the relationship between achieving transfusion independence and reduction in BMF in MF patients treated with pacritinib 200 mg twice daily (BID) vs ruxolitinib (RUX) on the phase 3 PERSIST-2 study. Methods: PERSIST-2 enrolled patients with platelet counts ≤100 x 10 9/L. This analysis focused on pacritinib 200 mg BID and on patients who received RUX as best available therapy (BAT) who enrolled ≥12 weeks prior to study termination and who required red blood cell (RBC) transfusions at baseline. The proportion of patients who achieved transfusion independence response (TI-R, any 12-week interval with no RBC transfusions) was ascertained for pacritinib vs RUX. The proportion of patients with BMF reduction (≥1 grade decrease in reticulin fibrosis from baseline at week 24) was reported among patients on pacritinib achieving TI-R vs. non-response (NR). Results: The analysis included 41 patients on pacritinib (median dose intensity 100% through week 24) and 18 on RUX (median daily dose 10mg at week 24). Baseline characteristics were similar between the groups, including median platelet count (41 vs 38 x 10 9/L) and median hemoglobin (8.7 vs 8.6 g/dL). All patients required RBC transfusion at baseline. A significantly greater proportion of patients treated with pacritinib vs RUX achieved TI through week 24: 37% (n=15/41) vs 6% (n=1/18), P=0.023. Nominally, this trend held for those with baseline platelets <50 x 10 9/L: 28% vs 8%, P=0.222. In addition, a greater percentage achieved a 50% reduction in RBC transfusions over any 12 weeks (49% vs 6%, P=0.001). Paired bone marrow assessments at baseline and week 24 were available for 18/41 of patients on pacritinib, of whom 44% (8/18) achieved TI-R on study. The proportion of patients who experienced BMF reduction (>=1 grade at any point) was significantly greater among TI-R (62.5%, n=5/8) compared to TI-NR (10%, n=1/10) on pacritinib ( P=0.043). Of the 5 patients who achieved TI-R and BMF reduction, all had grade 2-3 fibrosis at baseline, and 2 experienced a reduction from grade 3 to grade 1 ( Figure 1). By contrast, paired bone marrow biopsies were available for 5 patients on RUX, and there was no association between fibrosis reduction in TI-R (0%, n=0/1) and TI-NR (25%, n=1/4). Conclusions: In cytopenic MF patients from PERSIST-2, TI-R on pacritinib was associated with BMF improvement. Though these results are based on a small sample size, they contrast with recent data suggesting no correlation between BMF reduction and TI-R on the JAK1/2 inhibitors momelotinib and ruxolitinib ( Oh ST, et al. Blood 2022;140 (Supp 1):821-23). While differences in study design could have impacted these results, these findings suggest that distinct inhibitory profiles may further distinguish the clinical impact of these treatments. Further studies are warranted to confirm the relationship between BMF reduction and anemia benefit in patients treated with pacritinib. Study funded by CTI BioPharma Corp., a Sobi company

MSC Reprogramming and Aberrant Inflammatory Signaling in Myelofibrosis: Insights from Single-Cell RNA-Sequencing of Trephine Bone Marrow Cells

Myelofibrosis (MF) results from mutation-induced activation of JAK/STAT signaling in hematopoietic stem, progenitor and mature cells, and features a progressive, fibrosing stromal reaction, splenomegaly, constitutional symptoms and reduced life expectancy. Identification of disrupted cellular pathways within bone marrow (BM) is pivotal for therapeutic breakthrough, but fibrosis severely limits access to stromal elements for single-cell analysis. To overcome this, we utilized collagenase digestion of fresh BM trephine biopsy material surplus to diagnostic needs and profiled isolated cells using scRNAseq. 9 samples were obtained from 8 patients with MF (4, JAK inhibitor naïve; 4, on ruxolitinib); 4 samples were from 4 patients with MF 4, 10, 12 & 12 months after allogeneic transplant; and 7 samples (termed “normal controls”) were from 6 patients who had received an allograft for myelodysplasia or acute leukemia, and 1 who had received CAR-T cells for B-ALL. Normal controls exhibited normal or near-normal blood counts and lacked BM fibrosis. Each sample was subjected to collagenase digestion and flow sorted to isolate non-erythroid hematopoietic (CD45+GlyA-) and microenvironmental (CD45+GlyA-CD71-) cells for single cell RNA sequencing and downstream comparative analysis. 105,231 transcriptomes met quality control criteria (median 4975/sample, range 1036-13277, no significant difference between sample categories) and 43 transcriptional clusters were identified. Compositional analysis identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. While transcriptomes of ECs did not differ substantially, there were extensive transcriptional differences between MSCs recovered from MF vs. normal control samples. Subclustering revealed two subsets, MSC1 & MSC2 asymmetrically distributed between MF and normal controls. Intriguingly, MSCs recovered from MF patients after allograft occupied an intermediate position in principal component space indicating that reversion of disease-related MSC transcriptional reprogramming post-allograft occurs over a timescale of months. The reprogrammed transcriptional state of MF MSCs is defined by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Transcriptional change in MSCs correlated strongly with histopathological assessment of BM reticulin. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14 + monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls - the largest reported to date - reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.

Cardiovascular Risk Factors Are Common in Myeloproliferative Neoplasms and Portend Worse Survival and Thrombotic Outcomes

DISCUSSION Myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) are characterized by increased risk of arterial and venous thrombosis. Cardiovascular risk factors (CV RFs) including hypertension, hyperlipidemia, diabetes, smoking, and obesity likely contribute to thrombotic risk, but the exact incidence of these risk factors and the impact of CV RF modification in MPNs is less clear. The purpose of this study was to determine the prevalence of baseline CV RFs in MPN patients, investigate their association with genomic profiles, and evaluate their effect on long-term outcomes. We retrospectively analyzed patients who received targeted gene sequencing at Massachusetts General Brigham / Dana Farber Cancer Institute (N=977) from 2014-2023, and met WHO 2016 criteria for PV, ET, MF, or pre-fibrotic MF. CV RFs were identified through ICD-9 or 10 codes present prior to MPN diagnosis, and defined as hypertension, hyperlipidemia, diabetes, current smoking status, or BMI>30. Patient and treatment characteristics were described with summary statistics. Genomic profiles were compared between ET, PV, and MF patients with vs without a CV RF. Primary outcome was overall death. Secondary outcomes were venous thromboembolism, arterial thrombosis (including myocardial infarction and stroke), and transformation to MF or acute myeloid leukemia (AML). We calculated cumulative incidence functions of arterial/venous thrombosis as well as overall survival in patients with or without a CV RF. Hazard ratios (HR) were estimated for outcomes using Cox proportional hazards regression. Our cohort contained 399 (39.6%) ET, 312 (31.0%) PV, and 237 (23.5%) MF or pre-fibrotic MF patients. The median age at diagnosis was 58.5 years, and 47.9% of patients were male. The overall prevalence of hyperlipidemia, hypertension, and diabetes at MPN diagnosis was 16%, 20%, and 8%. The average BMI at diagnosis in all MPNs was 27.28, with 64% and 23% of patients having a BMI of >25 and >30. Six percent of MPN patients were current smokers at time of diagnosis, compared to 39% former smokers and 56% never smokers. ET and PV patients with ≥1 CV RF at MPN (N = 234, 32.9%) diagnosis were older (mean age 61.1 vs 52.1 years, p<0.001), and more likely to be male (50.9% vs 41.3%, p=0.02), non-White (12.4% vs 7.6%, p=0.015), and have a prior history of atherosclerotic disease (16.2% vs 4.4%, p<0.001), thrombosis (13.7% vs 5.0%, p<0.001), and heart failure (2.1% vs 0.4%, p=0.042). MF patients with ≥1 CV RF (N = 120, 50.6%) were also more likely to be older (mean age 67.5 vs 60.4 years, p<0.001), male (66.7% vs 53.9%, p=0.047), and have a prior history of atherosclerotic disease (19.2% vs 6.0%, p=0.003) (Table). Results of targeted gene sequencing closest to MPN diagnosis were analyzed. A similar proportion of driver ( JAK2, CALR, MPL) and concomitant ( TET2, ASXL1, DNMT3A, SRSF2, SF3B1, U2AF1, ZRSR2) mutations were seen in MPN patients with or without a CV RF. However, patients with ET or PV with ≥1 CV RF had a lower variant allele fraction (VAF) of their driver mutation (mean 41.0% vs 48.1%, p = 0.004), which was primarily driven by JAK2 (mean 43.5% vs 52.0%, p=0.003). In ET and PV, after adjusting for variables significant on univariate analysis, the presence of ≥1 CV RF was associated with higher risk of death from any cause (HR 1.73, 95% CI 1.08-2.76) and arterial thrombosis (HR 2.33, 95% CI 1.22-4.42). Among patients with MF, the presence of ≥1 CV RF was not associated with increased risk of death when adjusted for age, sex, and prior thrombosis (HR 1.36, 95% CI 0.84 - 2.20). MPN patients with ≥1 CV RF did not have increased rates of MF or leukemia progression (Fig). In our study, CV RFs are common among patients with MPN. CV RFs were associated with adverse outcomes, including death and thrombosis among patients with ET or PV. We found no differences in the molecular profiles in MPN patients with or without CV RFs, although a lower JAK2 VAF was seen in patients without CV RFs, which will need to be explored further. MPN patients with a CV RF had significantly worse overall survival and cumulative arterial thrombosis rates, although the presence of CV RFs does not impact MF or leukemia progression. However, our results highlight the importance of addressing CV RFs in MPN care to improve morbidity and mortality.

Use of Combination Therapies Including the XPO1 Inhibitor Selinexor in Is a Potential Effective Therapeutic Strategy to Treat Myelofibrosis Patients

JAK1/2 inhibitor therapy reduces the degree of splenomegaly and improves systemic symptoms of myelofibrosis (MF) patients, but does not delay disease progression and modestly prolongs overall survival. HDM2 antagonists (nutlins) have been reported to selectively eliminate MF CD34 + cells by activating the p53 pathway. Nutlins alone target malignant cells with wild type (WT) p53 and in clinical trials, HDM2 antagonists have led to reductions in spleen size, improvement of systemic symptoms and reduction in MPN driver mutation allelic burdens (Lu et al, Blood.2012; Mascarenhas et al, Blood. 2019). Although most patients with MF have WT TP53, mutations are present in a limited number of such patients and are associated with poor outcomes. We have searched for combinations of drugs to serve as a more effective and tolerable MF stem cell depleting therapy. Exportin 1 (XPO1) mediates the nuclear export of various proteins and RNAs (Azmi, et al. Nat Rev Clin Oncol. 2021) including tumour-suppressor proteins and growth regulators, such as RB1, p53, p63, p73, p21, cyclin B1 or D1 (Azmi, 2021). XPO1 is frequently overexpressed and/or mutated in human cancers and functions as an oncogenic driver. Suppression of XPO1-mediated nuclear export has been shown to be a potential therapeutic strategy to treat MF patients (Yan, et al. Clin Cancer Res. 2019). Selinexor (SEL) is an investigational, oral XPO1 inhibitor that may inhibit multiple pathways relevant in MF including JAK/STAT, ERK, and AKT (Tantravahi, ASH 2021). In the current study, we tested the effects of SEL on apoptosis, and cell cycle arrest in JAK2V617F cells with either TP53WT or TP53mut. We also evaluated the ability of SEL alone to eliminate MF CD34 + cells and in combination with other candidate drugs such as HDM201 (HDM2 antagonist), ruxolitinib (RUX), pan-BET inhibitor (JQ1) or a Bcl-2/Bcl-xL inhibitor (navitoclax). Treatment with SEL alone induced apoptosis of both TP53WT (UKE-1) and TP53mut (SET-2) MPN CD34 + cell lines in a dose-dependent fashion. The transcript levels of TP53 as well as its downstream genes, such as p21, PUMA, NOXA (intrinsic apoptotic pathway) and DR5 and Trail (extrinsic apoptotic pathway) were increased by 2-3 fold after SEL treatment of TP53WT and TP53mut cells. Importantly, SEL also increased TP63 and TP73 by ~2 fold but decreased the transcript levels of the anti-apoptotic Bcl-xL and c-Myc (by 20% and 50%), TP63 and TP73 are TP53 family members which promote p53 downstream events independently of WT p53. In addition, treatment with SEL for 2 days increased the proportion of cells in sub-G0/G1 phase from 16% (control) to 60% in UKE-1 cells and from 15% (control) to 53% in SET-2 cells. SEL and HDM201 alone induced apoptosis of primary MF but not healthy donor (HD) CD34 + cells in a dose dependent fashion. The combination of SEL with suboptimal doses of either HDM201, RUX, JQ1 or navitoclax showed additive pro-apoptotic effects on MF CD34 + cells. Colony formation assays showed that SEL, HDM201 and navitoclax alone decreased CFU-GM colony numbers generated by MF CD34 + cells in a dose-dependent fashion, yet had no effects on HD CD34 + cells, while RUX reduced both MF and ND colony formation. Combination treatment with SEL and 50nM of HDM201 or RUX led to a greater degree of reduction of MF CFU-GM colony numbers, but only SEL+HDM201 did not affect HD CFU-GM colony numbers (Figure 1). Furthermore, JAK2 genotyping showed that treatment with SEL, Rux and HDM201 alone decreased the absolute number of JAK2V167F+ colonies. Furthermore, SEL+HDM201 and SEL+RUX decreased the number of JAK2V617F+ colonies to greater extent than either drug alone, but SEL+HDM201 treatment led to the persistence of a greater numbers of JAK2WT colonies than SEL+RUX (Table 1). SEL+navitoclax did not further decrease JAK2V167F+ colonies than SEL alone (data not showed). These data suggested that SEL induces apoptosis and inhibits MPN CD34 + cell proliferation by affecting both p53-dependent and -independent pathways. SEL alone induces apoptosis and inhibits MF progenitor cells but not HD cells. The effects of SEL combined with either RUX or an HDM2 antagonist more effectively depleted mutated MF CD34+ cells than either drug alone. The combination of SEL+HDM201 spared the reservoir of WT progenitors, and reduced JAK2 mutated progenitor cells to a great degree suggesting that this combination might be associated with less hematological toxicity and greater efficacy.

ALK2 and JAK2 Inhibition for Improved Treatment of Anemia in Myelofibrosis Patients: Preclinical Profile of an ALK2 Inhibitor Zilurgisertib in Combination with Ruxolitinib

Introduction: Anemia is a common occurrence in patients with myelofibrosis (MF) and is associated with the need for red blood cell transfusion and poor clinical prognosis. JAK inhibitors such as ruxolitinib are used extensively to treat symptoms of MF and improve quality of life and overall survival. However, JAK inhibitors may also contribute to myelosuppression. Therapeutic interventions that allow for optimization of JAK inhibition with no concern of anemia would benefit patients with MF and potentially lower the rate of treatment discontinuation or suboptimal dosing. Recent studies have demonstrated that inhibition of ACVR1/ALK2, a bone morphogenetic protein receptor that is upstream of the transcriptional regulation of hepcidin, could reduce serum hepcidin levels in patients with MF and improve anemia (Verstovsek S et al. Lancet 2023; Oh S et al. Clin Lymphoma Myeloma Leuk 2022). Reducing levels of hepcidin, a key regulator of plasma iron levels, and restoring erythropoiesis would broadly benefit patients with MF. Based on these recent data, we developed zilurgisertib, a potent and selective ALK2 inhibitor that could be administered in combination with ruxolitinib at doses titrated to the needs of patients with MF. Methods and Results: In biochemical and cellular assays, zilurgisertib inhibited ALK2 kinase activity and SMAD1/5 phosphorylation with IC 50 values of 15 nM and 63 nM, respectively. In Huh-7 cells stimulated with BMP-6, zilurgisertib inhibited hepcidin production with an IC 50 of 20 nM, demonstrating the compound is a potent ALK2 inhibitor capable of regulating iron homeostasis via hepcidin. To assess possible off-target effects of zilurgisertib, kinome profiling at 10 μM ATP was performed at Reaction Biology (Malvern, PA) to determine the overall specificity across 356 kinases (see Figure). At 200 nM, zilurgisertib only inhibited ALK2, ALK1 (to 50%), and ALK6 (to 48%). In addition, zilurgisertib at 20 μM did not affect viability of HEK293 cells, a human cell line commonly used to assess general cell health and compound toxicity. Similarly, zilurgisertib did not affect viability of human fibroblasts or endothelial cells at concentrations up to 5 μM. To test whether the combination of ruxolitinib and zilurgisertib could suppress hepcidin and restore erythropoiesis in an in vivo mouse model of cancer-induced anemia, B16F10 cells were injected intraperitoneally, mimicking a metastatic tumor that leads to anemia 1 week after injection. In a dose-dependent manner, zilurgisertib improved hemoglobin by 2 to 3 g/dL and red blood cell counts, while reducing both liver pSMAD and circulating hepcidin levels by 50% or more compared with vehicle control. The combination of zilurgisertib with ruxolitinib had no effect on this activity, suggesting that inhibition of JAK2 does not inhibit erythropoiesis restored following ALK2 inhibition. Conclusion: Taken together, the potent and selective on-target activity of zilurgisertib suggests that ALK2 inhibition could reduce hepcidin and improve anemia, and that the combination of zilurgisertib with ruxolitinib is a rational and attractive approach to mitigate anemia in patients with MF. The combination of ruxolitinib and zilurgisertib is currently being evaluated in a phase 1 clinical trial in patients with anemia due to myeloproliferative disorders (NCT04455841).

Phase 1/2 Study of TP-3654, a Selective PIM1 Kinase Inhibitor: Preliminary Data Showed Clinical Activity and Cytokine Reductions in Relapsed/Refractory Myelofibrosis Patients

Background: PIM1 expression is significantly upregulated in hematologic malignancies including myelofibrosis (MF) and has been shown to play a role in modulating the activity of cytokine induced PI3K/AKT and JAK/STAT pathways. The distinct clinical features of MF are partly attributed to the elevated circulating cytokines; a subset of which are shown to correlate with poor prognosis. TP-3654, an oral investigational highly selective PIM1 kinase inhibitor, is currently being evaluated in a phase 1/2 study in patients (pts) with relapsed/refractory MF. In preclinical studies, TP-3654 alone and in combination with ruxolitinib showed spleen size and bone marrow (BM) fibrosis reduction in murine MF models. Importantly, TP-3654 also showed reduction of cytokine response genes and serum TGF-β in progenitor cells and JAK2V617F murine MF model, respectively. Methods: This phase 1/2 study evaluates the safety and efficacy of TP-3654 monotherapy in pts with MF (NCT04176198). Key eligibility criteria include primary or secondary MF; previously treated with or ineligible for JAK inhibitor treatment; DIPSS intermediate 1, 2 or high-risk MF; platelet ≥25x10 9/L; splenomegaly (≥450 cm 3 by imaging); and ≥2 measurable symptoms per MFSAF v4. The study aims to identify the MTD and/or RP2D of TP-3654 monotherapy and to assess safety, clinical activity (spleen volume reduction [SVR], total symptom score [TSS] improvement), PK, and PD markers (cytokine profile, BM fibrosis etc.). We investigated the patterns of similarity of 43 cytokines changes after 12 weeks of TP-3654 treatment using principal component analysis (PCA). Results: As of 20 June 2023, a total of 23 pts enrolled across 5 dose levels in the phase 1 dose escalation part. At baseline, median age 73 years (range 61, 80), spleen volume 2008.2 cm 3 (range 609, 6006), TSS 20.8 (range 4, 62), platelet 115 x10 9/L (range 64, 520), hemoglobin 10.1 g/dL (range 5.9, 13.7; 5 pts requiring transfusion). All pts, except one, had received ≥1 prior JAK inhibitor treatment (median 12.9 months [range 2.3, 81.0]). Median duration of TP-3654 treatment was 14.9 weeks (range 2, 96). No DLT occurred. Treatment-related adverse events (TRAEs) occurring in ≥20% of pts included grade 1/2 nausea, vomiting, and diarrhea. Grade 3 TRAEs were diarrhea (n=1) and platelet count decreased (n=2). Mean hemoglobin and platelet counts remained stable throughout the 24-week treatment period. SVR observed in 10 of 13 evaluable pts treated for ≥12 weeks (median best change -11%, range +97% to -63%); 3 pts showed ≥35% SVR. TSS improvement were observed in 12 of 13 evaluable pts (median best change -70%, range +9 to -100%); 7 pts showed ≥50% TSS response (Fig 1A); 5 pts had durable response for ≥12 weeks. Broad reductions in cytokines observed as early as within the first 24 hours after TP-3654 treatment. At week 12, pts with higher cytokine reductions correlated with greater TSS improvement. Two components of the PCA, PC1 and PC7, include MF associated cytokines such as IL-8, IL-18, CD40, ENRAGE amongst others, were reduced with TP-3654 treatment and these reductions correlated with symptom improvement (Fig 1B). BM fibrosis was reduced in one pt who also achieved spleen and symptom responses, showed MF associated cytokine reductions, and is on active treatment for 2 years. Conclusions: This preliminary data of TP-3654 in relapsed/refractory MF pts showed early signs of clinical activity including spleen volume reduction, TSS improvement, and correlating cytokine reductions. TP-3654 is well tolerated with limited myelosuppressive adverse events. Enrollment is ongoing as monotherapy and current data support the development of TP-3654 in combination with JAK inhibitors given the preliminary clinical activity and minimal cytopenia.

Fedratinib Treatment Reduces the Inflammatory Cytokine Profile and Decreases Exhausted T Cells Correlating with Clinical Response in Patients with Myelofibrosis: Biomarker Analysis from the Phase 3 FREEDOM2 Trial

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by a hyperactive JAK-STAT pathway, splenomegaly, and constitutional symptoms. JAK inhibitors (JAKi) have been the mainstay in MF treatment over the past decade. Pro-inflammatory cytokines, the levels of which have prognostic value (Tefferi et al. J Clin Oncol 2011), have been shown to be upregulated in MF. However, there is paucity of data on chronic immune dysregulation in pathogenesis of MF. Here, we report the multiplatform biomarker analysis including immune changes from phase 3 (FREEDOM2) trial in patients with MF previously exposed to ruxolitinib (Rux) and treated with fedratinib vs. best available therapy (BAT) (primary efficacy: 35.8% vs 6% responders at the end of cycle 6 (EOC6) in fedratinib vs BAT, respectively). Of note, 78% of the patients on BAT continued RUX therapy until EOC6, allowing direct comparison between RUX and fedratinib. Methods: In the FREEDOM2 trial, patients (N=201) were randomized into two arms, fedratinib (400 mg/day) or BAT in a 2:1 ratio, respectively. The primary endpoint was the proportion of patients with ≥35% reduction of spleen volume (SVR35) on MRI at the end of cycle 6 (EOC6). In the FREEDOM2 biomarker cohort, mutational analysis was performed in enriched CD66b+ cells using the MLL myeloid panel targeting 74 genes. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels from C1D1 to EOC6. Custom Q2 immune panels to detect myeloid and lymphoid subsets were used for immunophenotyping of peripheral blood samples. Results: Baseline mutations in FREEDOM2 were commonly found MPN mutations that include JAK2 V617F (71%), ASXL1 (43%), CALR (22%), TET2 (22%) and MPL (11%). However, baseline mutation status was not a predictor of response. Patients receiving fedratinib demonstrated significant down-regulation of several pro-inflammatory cytokines including CRP, ENRAGE, IL-18, IL-16, TIMP-1, and increase of anti-inflammatory cytokines including adiponectin, MMP2, and CEA (Wilcoxon signed rank test, BH adjusted p <0.001) at EOC6 vs C1D1. In addition, the change in levels of many cytokines correlated with the clinical endpoint (SVR35) demonstrating that the therapeutically relevant changes were mediated by fedratinib (Pearson rho P<0.05) (Figure 1). Importantly, fedratinib was superior to the BAT in its ability to alter levels of key cytokines (p <0.05) at EOC6. PD-1 expression has been shown to be upregulated in CD4+ and CD8+ T cells in MF patients and is associated with poor overall survival. However, RUX did not have a significant effect on PD1+ T cells (Veletic et al. Haematologica 2021). Interestingly, flow cytometric analysis revealed that fedratinib decreased exhausted CD8+ T cells (PD1+) up to 40% (∆ median change -11.4%,p<0.001)) (figure 2). Importantly, the reduction in exhausted T cells correlated with the reduction in spleen volume with fedratinib treatment ( R= -0.51, P=0.00011; Figure 2). Consistent with this, we also observed median increases in NK (+3%, p<0.001) and NK-T (+13.8%, p<0.001) populations when compared to the BAT arm. Conclusions: To our knowledge, this is the first comprehensive study detailing cytokine and immune changes in patients with MF in a clinical trial. In addition, our data for the first time demonstrates the impact of fedratinib on the immune system and its ability to reduce exhausted T cells, highlighting the immune modulatory mechanism of fedratinib. Notably, the cytokine and immune changes correlated with the primary endpoint and were not observed in the BAT arm demonstrating superior efficacy of fedratinib in previously RUX exposed patients. Furthermore, since majority of the patients in the BAT arm continued to receive RUX, these findings highlight the mechanistic differences between fedratinib and RUX on clinical response in MF (SVR35). Overall, our data highlight multiple novel avenues to achieve therapeutic efficacy in MF and positions fedratinib as an effective therapy with a distinctive mechanism of action. Additional analyses on the effect of fedratinib treatment on myeloid immune subsets and transcriptomic changes are underway to gain comprehensive mechanistic understanding.

An Analysis of Ruxolitinib Dosing for Myelofibrosis in Real-World Practice

Background: Myelofibrosis (MF) is a myeloid neoplasm characterized by splenomegaly, constitutional symptoms, and cytopenias. Notably, worsening cytopenias in MF patients is directly correlated with poor survival ( Coltro, et al. Blood Cancer J. 2022;12(8):116). The JAK1/2 inhibitor ruxolitinib (RUX) is effective in decreasing splenomegaly and improving symptom control at starting doses of 30-40 mg total daily dose (TDD), though drug-induced cytopenias often lead to significant dose reductions. Doses of ≤20 mg TDD are associated with decreased efficacy for spleen response, and doses of ≤10 mg TDD demonstrate minimal efficacy for spleen or symptom reduction ( Verstovsek, et al. Onco Targets Ther. 2014;7:13-21). Treating with less clinically effective doses may not be optimal in light of newer, less myelosuppressive JAK2 inhibitors that can be administered without dose reduction, regardless of baseline cytopenias. This study describes RUX dosing patterns in real-world community practice. Methods: This retrospective, observational study included deidentified data of adult patients from the IntegraConnect PrecisionQ database (~80% community practice) with MF treated with first-line RUX and with ≥2 office visits (with a criterion of 2 MF diagnosis codes reported within 180 days) from January 2016 to July 2022. Patients were stratified by baseline platelet count (PLT). Data on RUX TDD at initiation and at first dose modification were collected, with a focus on less clinically effective dosing of ≤20 mg TDD and ≤10 mg TDD. Results: Of 129 patients on RUX with a median age of 75 (range: 42-89) years, 61% (n=79) had PLT >200x10 9/L, 19% (n=24) had PLT 100-200x10 9/L, and 20% (n=26) had PLT <100x10 9/L. Across all PLT strata, 88% (n=114) were initiated on RUX at a TDD of ≤20 mg, and 51% (n=66) were started at a TDD ≤10 mg. Nearly half of patients with higher baseline platelet counts (>100 x 10 9/L) were started on doses ≤10 mg TDD (Fig 1). Among 79 patients with PLT >200x10 9/L, starting RUX TDD was ≤20 mg in 87% (n=69) and ≤10 mg in 43% (n=34). A total of 49 patients underwent subsequent dose modification (median time [min, max]: 47 days [10, 1336], with 19 dose increases vs 30 dose decreases. Of patients whose dose increased, only 4 escalated to a TDD >20 mg. Despite dose modification, RUX TDD remained ≤20 mg in 90% (44/49) and ≤10 mg in 43% (21/49) of patients. Similar to patients with baseline PLT >200x10 9/L, the majority of patients with PLT 100-200x10 9/L started on TDD ≤20 mg (87.5% [21/24]), with a high proportion on ≤10 mg (50% [12/24]). Among the 103 patients with PLT >100x10 9/L for whom a recommended starting dose is provided in the RUX package insert, 88% (91/103) initiated RUX below the recommended dose. Of the 61 patients who underwent subsequent dose modification (median time: 47 days [10, 1336], only 10% (6/61) were escalated to a dose recognized as clinically effective. Conclusions: These real-world data suggest that a majority of patients starting RUX, including those with PLT >200x10 9/L, are initiated on ≤20 mg TDD, and the majority of patients are not titrated up to the doses tested in the pivotal studies that led to RUX approval. These data suggest that clinicians are hesitant to prescribe RUX at doses known to be clinically effective. Together with the timing of dose modification, this suggests that there may be concerns related to toxicity or treatment-related cytopenias. Recognizing that JAK2 inhibition remains an important part of managing MF for patients, clinicians now have alternative treatment options. These newer treatments inhibit additional pathways (IRAK1/ACVR1), are less myelosuppressive, and can maintain dose intensity regardless of baseline platelet count. Study funded by CTI BioPharma Corp., a Sobi Company.

Rker-050, a Modified Activin Receptor Type Iia Ligand Trap, Promoted Erythropoiesis in a Murine Model of Myelofibrosis

Myelofibrosis (MF) is one of several myeloproliferative neoplasms most often associated with anemia due to ineffective hematopoiesis, inflammation, aberrant bone metabolism and progressive fibrosis in the bone marrow (BM). As a compensatory mechanism, extramedullary hematopoiesis is initiated in the spleen, resulting in splenomegaly. Megakaryocyte hyperplasia/dysplasia are thought to be one of the main drivers of MF disease progression. Evidence also suggests that dysregulated TGF-β superfamily activity contributes to ineffective hematopoiesis. KER-050 and its research form, RKER-050, are investigational modified activin receptor type IIA ligand traps designed to inhibit specific TGF-β superfamily ligands, including activin A, activin B, growth and differentiation factor (GDF) 8 and GDF11, to promote erythropoiesis and thrombopoiesis. Additionally, the KER-050 target ligands promoted fibrosis and bone resorption in preclinical studies. Therefore, by inhibiting these ligands, KER-050 could potentially rebalance the bone marrow microenvironment, including reducing fibrosis, allowing restoration of hematopoiesis and alleviating extramedullary hematopoiesis and associated splenomegaly. To investigate the potential of RKER-050 to reverse ineffective hematopoiesis in MF, a hypomorphic Gata1 MF mouse model (Gata1Low; MF mice) was utilized which presents similar characteristics to human MF, including ineffective hematopoiesis, cytopenias, extramedullary hematopoiesis, defective bone deposition and BM fibrosis. this study, we also observed aberrant bone growth in the cortex of the hind limb long bones which inappropriately extended into the bone marrow, resulting in limited BM compartment space and demonstrating a severe disease state in these mice. Anemia status of MF mice was confirmed at the start of the treatment. MF mice showed a 16% reduction in hemoglobin (Hgb) compared to WT control mice. After 12 weeks of treatment, MF+VEH mice continued to exhibit a significant or trending decrease in red blood cells (RBCs; -19%), Hgb (-20%), and hematocrit (Hct; -10%) compared to WT mice. In contrast, MF+050 mice had a significant recovery of RBCs (+31%), Hgb (+24%) and Hct (+20%), compared to MF+VEH mice, demonstrating that RKER-050 reversed anemia in this advanced disease MF model. To assess the mechanism of action of RKER-050, we evaluated erythroid precursor populations in the BM. While MF+VEH mice showed a significant reduction in erythroid progenitors compared to WT mice, These data support increased BM erythropoiesis with RKER-050 treatment. In addition, we evaluated the effect of RKER-050 on thrombopoiesis. MF+VEH mice were severely thrombocytopenic and had significantly increased megakaryocyte progenitors (MkP; +591%) compared to WT mice. Although RKER-050 significantly reduced MkP (-33%; MF+050), suggesting RKER-050 may positively influence the megakaryocyte lineage, this did not translate to improvements in thrombocytopenia, and platelet counts remained similar to MF+VEH. Additional data will be presented that investigates the potential of RKER-050 treatment in (6-month vs 10-month-old mice, where disease is less severe) to further evaluate the potential to impact cytopenias, extramedullary hematopoiesis and BM fibrosis. Overall, in the context of severe disease, our results suggest RKER-050 can promote erythropoiesis and reverse anemia in the BM in by rebalancing hematopoiesis in the BM niche. Reestablishing BM hematopoiesis could obviate the need for compensatory extramedullary hematopoiesis in the spleen, the major driver of splenomegaly in MF patients. In conclusion, KER-050 represents a potentially promising approach for patients with MF and other hematological diseases where ineffective hematopoiesis occurs.

Selinexor Plus Ruxolitinib in JAK Inhibitor (JAKi)-Naïve Patients with Myelofibrosis: Long Term Follow up from XPORT-MF-034 Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by splenomegaly, constitutional symptoms, and inflammation due to unregulated, clonal proliferation of hematopoietic stem cells in the bone marrow. Common genetic drivers include JAK2, CALR, and MPL. Although improvements in splenomegaly and symptoms occur with the current approved therapies, additional assessments are needed to understand the impact of therapies on disease modification. Increased burden of variant allele frequency (VAF) of driver mutations is associated with poor disease outcomes, and increased expression of pro-inflammatory cytokines is implicated in disease pathophysiology and symptoms burden. Selinexor (SEL) is an investigational oral XPO1 inhibitor that may inhibit multiple MF-relevant pathways including STAT, ERK, and AKT. Preclinical studies have shown potential synergy between SEL and ruxolitinib (RUX) treatment in vivo. Safety and efficacy analysis of SEL plus RUX in JAKi treatment-naïve MF in a Phase 1 study supported a 60 mg SEL dose as the recommended dose in a Phase 3 study. Here we include exploratory analyses of a range of biomarkers in the Phase 1 study of SEL and RUX in JAKi-naïve myelofibrosis. Methods: Phase 1 of XPORT-MF-034 (NCT04562389) is an open-label study evaluating the safety and efficacy of SEL at 40mg and 60mg once weekly plus RUX with JAKi-naïve MF. Assessments included safety and rates of spleen volume reduction of ≥35% (SVR35) and total symptom score reduction of ≥50% (TSS50). Platelet and hemoglobin levels were also assessed. Longitudinal clinical biomarker assessments included %VAF change at week 24 for driver genes and plasma cytokine levels at week 4 (Cycle 2 Day 1). Bone marrow (BM) biopsies were obtained at screening and week 24, then evaluated for bone marrow fibrosis (BMF) and other BM cellular markers (CD61, CD71). Results: As of April 10, 2023, a total of 24 patients (pts) received at least one dose of SEL (40 mg: n=10; 60 mg n=14). The most common adverse events (AEs) overall of any grade were nausea (75% majority grade 1-2), fatigue (58%), anemia (54%), and thrombocytopenia (54%). Those receiving a prophylactic anti-emetic reported lower incidence and severity of nausea AEs compared to those that did not; pts experienced a median weight increase of 3.3 kg at week 24. Two pts discontinued treatment due to treatment-related AEs (thrombocytopenia and neuropathy). Generally stable hemoglobin levels were observed in 48% of transfusion-independent pts. Median hemoglobin levels were 10.1 g/dL at baseline, 9.3 g/dL at week 12, and 10.2 g/dL at week 24. Median platelet counts remained generally stable with median values of 258 k/uL at baseline, 164 k/uL at week 12, and 193 k/uL at week 24. SVR35 at week 24 in the intent-to-treat (ITT) population was achieved by 40% of the 40 mg cohort and 79% of the 60 mg cohort and TSS50 was achieved by 10% and 58%, respectively. Mutation analysis showed no pts were triple negative, with 18 harboring JAK2 mutations, 5 with CALR, and 1 with MPL. Of 13 pts with available data at week 24, 5 pts (38%) had a reduction of VAF that was ≥20%, and 3/5 of these pts had high VAF (>50%) driver mutations at baseline and were also characterized as HMR. A panel of 74 cytokines was evaluated in 21 MF pts with baseline and week 2 samples (including 10 with end-of-treatment [EOT] samples), and in 10 healthy donors. Comparing baseline MF to healthy samples, 50% of the analyzed cytokines were elevated. A rapid and durable decrease of pro-inflammatory MF-relevant cytokines including TGF-β1, IFNɣ, TNF-α, IL-7, IL-1Ra, and IL-16 was observed in the majority of pts at week 2 and continued into EOT. Notably, the reduction in IL-18 correlated to SVR and TSS response at week 24, with R 2 values of 0.29 and 0.28, respectively. BM analysis at week 24 showed an increased number of erythroid CD71+ elements in 3/14 pts. Conclusions: SEL plus RUX was generally well tolerated and manageable.Evidence of potential disease modification in pts with JAKi-naïve MF patients was seen via rapid stabilization of platelets and maintaining hemoglobin levels, VAF reduction, and pro-inflammatory cytokine reduction. Promising biomarker and efficacy data suggests that SEL in combination with RUX has the potential to become a novel, first-line treatment for pts with MF. Updated clinical data with longer follow-ups and additional subgroup analyses will be available at the time of presentation.

A Global, Phase 3, Randomized, Double-Blind Study to Evaluate Safety and Efficacy of Selinexor, an XPO-1 Inhibitor, in Combination with Ruxolitinib in JAK Inhibitor-Naïve Myelofibrosis (XPORT-MF-034)

Background and Significance: Myelofibrosis (MF) is a myeloproliferative neoplasm that commonly harbors somatic gene driver mutations in Janus kinase 2 ( JAK2), calreticulin ( CALR), and myeloproliferative leukemia virus ( MPL) genes. Selinexor is an investigational oral XPO1 inhibitor that may inhibit MF-relevant pathways including STAT, extracellular signal-regulated kinase (ERK), protein kinase B (AKT) and p53. Furthermore, in vivo preclinical studies have shown potential synergy between selinexor and ruxolitinib treatment. The Phase 3 trial was initiated based on data from the Phase 1 portion of XPORT-MF-034 evaluating the combination of selinexor and ruxolitinib in JAKi-naïve patients with MF. Previously we reported that the combination of selinexor and ruxolitinib was generally tolerable and manageable and that the 60 mg selinexor dose cohort showed 71% and 79% of the intent-to-treat (ITT) population achieving a ≥35% reduction in spleen volume from baseline (SVR35) at Week 12 and Week 24, respectively; SVR35 rates were consistent across subgroups, including gender and regardless of ruxolitinib starting dose. Robust symptom improvement was also observed with 58% of the ITT achieving a ≥50% reduction in total symptom score from baseline (TSS50) at Week 24 (Ali et al., ASCO 2023; Ali et al., AACR 2023). These data provided strong support to further evaluate selinexor (60 mg) and ruxolitinib in patients with JAKi-naïve MF. Study Design and Methods: The XPORT-MF-034 (NCT04562389) trial includes a global, Phase 3 randomized, double-blind, placebo-controlled study designed to evaluate the combination of 60 mg selinexor and ruxolitinib. Patients will be randomized 2:1 to receive oral selinexor 60 mg or placebo once weekly (28-day cycle) and twice daily ruxolitinib. Select eligibility criteria include patients ≥18 years of age, with a spleen volume of ≥450 cm 3 by MRI or CT, dynamic international prognostic scoring system (DIPSS) of intermediate-1, intermediate-2, or high-risk, active symptoms of MF as determined by presence of at least 2 symptoms with a score ≥3 or total score of ≥10 at screening using the MFSAF v4.0, currently not eligible for stem cell transplantation, ECOG PS ≤2, platelet count ≥100 x 10 9/L, and creatinine clearance >15mL/min. Patients will be excluded if more than 10% blasts are present in peripheral blood or bone marrow; received previous treatment with JAK inhibitors for MF, or previous treatment with selinexor or other XPO1 inhibitors. The co-primary study endpoints are SVR35 at Week 24 as measured by MRI or CT scan and TSS50 at Week 24 as measured by the Myelofibrosis Symptom Assessment Form (MFSAF) V4.0. The key secondary endpoint is anemia response as per the International Working Group Myeloproliferative Neoplasms Research and Treatment and European Leukemia Network (IWG-MRT and ELN) criteria at Week 24. Results: TheXPORT-MF-034 Phase 3 trial is currently open for enrollment; a total of 306 JAKi-naïve MF patients will be enrolled and the study was initiated on June 28, 2023.

Population Pharmacokinetic and Pharmacodynamic Analysis of Navtemadlin in Patients with Relapsed and Refractory (R/R) Myelofibrosis (MF) and Other Myeloid or Solid Tumor Malignancies

Background:Murine double minute 2 (MDM2), a key negative regulator of the tumor suppressor protein p53, is overexpressed in malignancies such as MF and Merkel cell carcinoma (MCC). Navtemadlin is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis of TP53WT tumors. Navtemadlin has been tested in 21- or 28-day treatment cycles, with 5 or 7 consecutive days of dosing per cycle. Navtemadlin elicits plasma concentration-dependent increases in serum macrophage inhibitory cytokine-1 (MIC-1). An increase in MIC-1 relative to baseline MIC-1 serum concentration is a key pharmacodynamic (PD) marker of response to navtemadlin treatment (Zhang et al. Xenobiotica 2022). A population pharmacokinetic (PK) model was developed, based on patients with R/R MF (N=113, NCT03662126); MCC (N=35, NCT03787602); solid tumors and multiple myeloma (ST/MM N=105, Gluck et al. Invest New Drugs 2019); R/R acute myeloid leukemia (AML, N=35, Erba et al. Blood Adv 2019); and healthy volunteers (HV, N=30, Wong et al. Clin Pharmacol Drug Dev 2022).The objectives wereto characterize navtemadlin PK and covariate effects and compare steady-state (SS) PK and PD across cancer types. Methods:A two-compartment PK model with first-order absorption and elimination was estimated using NONMEM ® (first-order conditional estimation with interaction). Four transit compartments captured absorption. Inter-subject variability was modeled as lognormal. A stepwise covariate search used forward addition (p<0.05) followed by backward elimination (p<0.01). Log-transformed SS MIC-1 ratio to baseline (fold ratio) was tested in a fixed-effects linear regression model with respect to SS exposures, with baseline MIC-1 as the covariate. The final population PK and MIC-1 models were evaluated by inspection of parameter tables, by overlaying model predictions with observed data, and by standard residual diagnostics. Results:The PK estimation used 5,282 concentrations from 318 subjects, including 1,415 concentrations in 113 patients with R/R MF. Tumor type affected absorption rate (median, 0.635/hr in MCC; slower vs other types), apparent clearance (median, 8.90 L/hr in MCC; lower vs other types), and apparent central volume (median, 64.4 L in MCC; similar to AML, but lower vs other types) (Table 1). Accordingly, simulated SS area under the curve (AUCss) differed across populations in the order MCC>AML>MF>ST/MM = HV (Figure 1). Covariate analysis indicated median AUC increased with the inflammation marker C-reactive protein, by 21% at 90 th percentile CRP, and with age, by 5% at 90 th percentile age of 75 y, compared to the median, 66 y. Men had 24% lower relative bioavailability than women. Taking navtemadlin with food slowed absorption by 10% but had no impact on AUCss. Other tested covariates, including body weight, creatinine clearance, aspartate transaminase, alanine transaminase, bilirubin, albumin, alpha 1-acid glycoprotein, race, UGT1A1 genotype, recent ruxolitinib treatment in MF, and CYP3A4 inducer/inhibitor concomitant medications, were not significant. Median MIC-1 fold ratio increased with navtemadlin AUCss. Higher baseline MIC-1 resulted in lower SS MIC-1 fold ratio. Median baseline MIC-1 differed by tumor type. In R/R MF, baseline MIC-1 was 2,297 ng/L and SS fold ratio of MIC-1 was 7.79 (N=91). In MCC, baseline MIC-1 was 1,718 ng/L and SS fold ratio of MIC-1 was 16.7 (N=29). In R/R MF, higher baseline spleen volume was also associated with lower SS MIC-1 fold ratio. Exposure-MIC-1 relationships in R/R MF and MCC were generally similar to other tumor types and HV populations (Allard et al. Hemasphere 2019; Zhang et al. Xenobiotica 2022). Conclusions:Tumor type influenced navtemadlin exposure with simulated exposure in R/R MF patients at the selected Phase 3 dose of 240 mg comparable to exposures in MCC patients at 180 mg, the selected Phase 2 dose for that indication. Cancer-associated inflammation and/or treatment history, as well as age and female sex, may increase navtemadlin exposure. The PD marker MIC-1 responds to navtemadlin in a concentration-dependent fashion. The overall SS fold change of MIC-1 PD responses was lower in disease states or subjects with higher baseline MIC-1. These studies demonstrate potent, dose-dependent PK/PD responses following p53 activation with the novel MDM2 inhibitor, navtemadlin, across diverse tumor types.

TP53 mutations and Their Impact on Survival in Patients with Myeloproliferative Neoplasms

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by > 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p<0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

Outcomes after Bone Marrow Versus Peripheral Blood Stem Cell Matched Unrelated Donor Hematopoietic Stem Cell Transplantation

Background: Matched unrelated donors (MUD) have become an acceptable alternative donor for patients requiring potentially curative hematopoietic stem cell transplantation (HSCT) and lacking a matched sibling donor. Patients may undergo a bone marrow (BM) or peripheral blood stem cell (PBSC) graft, with previous studies showing similar survival outcomes. However, BM grafts have shown a lower incidence of chronic graft-versus-host disease (GVHD) but a higher relapse rate and slower engraftment. We aimed to compare the outcomes of BM versus PBSC in patients undergoing MUD HSCT. Methods: For this retrospective single-center study, we included all (n=276) adult MUD HSCT recipients at the University of Kansas Medical Center who had at least one year of follow-up (August 2016 to July 2021). We conducted a bivariate analysis using chi-squared and ANOVA tests. The Kaplan-Meier and regression analyses were also performed. Data analyses were conducted using SPSS version 28. Statistical significance was considered at p<0.05. Results: Our study included 276 MUD patients including 110 PBSC (40%) and 166 BM (60%) grafts. The median age was 55 (range 21-77) years, and 158 patients (57%) were males. Eighteen patients (6%) were ethnic minorities, and 89 (32%) patients had a Karnofsky performance status of ≥90%. 118 (43%) patients had a hematopoietic cell transplantation-specific comorbidity index of 3 or higher. Hematologic diseases included acute myeloid leukemia (n=119, 43%), myeloid disorders (n=77, 28%), acute lymphoblastic leukemia (ALL) (n=35, 13%), Lymphoma (n=15, 5%), and others (n=30, 11%). Sixty-two percent of patients were in complete remission at the time of transplantation, and patients not in remission included myelodysplastic syndrome, myelofibrosis, and lymphoma patients. Reduced-intensity conditioning (RIC, n=162, 58%) and myeloablative conditioning (MAC) (n=114, 41%) were used. The median follow-up was 55 (21-73) months. Overall survival (OS) was not reached in either BM or PBSC MUD HSCT group, while a median disease-free survival of 45 and 49 months was noted in PBSC and BM groups respectively (p=0.915). We observed similar rates of relapse (25% vs 27%, p=0.413), one-year overall survival (75% vs 72%, p=0.679), one-year disease-free survival (63% vs 62%, p=1.00), and one-year non-relapsed morality (NRM, 23% vs 21%, p=0.779) after PBSC or BM graft. Bone marrow graft recipients had delayed engraftment of neutrophils (20 days vs 16 days, p<0.001) and platelets (>20 K/uL: 24 days vs 16 days, p<0.001; >50 K/uL: 28 days vs 18 days, p=0.006) compared to PBSC recipients. Bone marrow graft recipients had similar rates of grade II-IV acute GVHD (50% vs 52%, p=0.714) and grade III-IV GVHD (12% vs 11%, p=0.847), but lower rates of 1-year chronic GVHD (39 % vs 55%, p=0.014) compared to PBSC recipients. In the subgroup analysis stratified by conditioning intensity, faster engraftment of neutrophils (median 16 vs 21 days, p<0.001) and platelets (>20 K/uL: 18 days vs 27 days, p<0.001; >50 K/uL: 18 days vs 33 days, p<0.001) was observed with PBSC as compared to BM graft in myeloablative transplant recipients, while there was no statistically significant association with the rates of acute or chronic GVHD, NRM, Relapse, DFS, and OS after a PBSC or BM graft. Among the RIC transplant recipients, faster neutrophil engraftment (mean 17 vs 20 days, p<0.001) and higher 1-year extensive chronic GVHD (52% vs 32%, p=0.015) were observed with PBSC compared to BM graft whereas no statistically significant difference was noted in platelet engraftment, acute GVHD, NRM, Relapse, DFS, and OS after a BM or PBSC graft. Conclusions: Allogeneic matched unrelated donor hematopoietic stem cell transplant recipients had similar rates of overall and disease-free survival, relapse, non-relapse mortality, and acute GVHD after a bone marrow or peripheral blood stem cell graft; however, delayed neutrophil and platelet engraftment with a lower incidence of chronic GVHD was noted after a bone marrow graft compared to peripheral blood stem cell graft.