Myeloproliferative Neoplasms Therapy Research Articles

Mutant Calreticulin in MPN: Mechanistic Insights and Therapeutic Implications.

More than a decade following the discovery of Calreticulin (CALR) mutations as drivers of myeloproliferative neoplasms (MPN), advances in the understanding of CALR-mutant MPN continue to emerge. Here, we summarize recent advances in mehanistic understanding and in targeted therapies for CALR-mutant MPN. Structural insights revealed that the mutant CALR-MPL complex is a tetramer and the mutant CALR C-terminus is exposed on the cell surface. Targeting mutant CALR utilizing antibodies is the leading therapeutic approach, while mutant CALR-directed vaccines are also in early clinical trials. Additionally, chimeric antigen receptor (CAR) T-cells directed against mutant CALR are under evaluation in preclinical models. Approaches addressing the cellular effects of mutant CALR beyond MPL-JAK-STAT activation, such as targeting the unfolded protein response, proteasome, and N-glycosylation pathways, have been tested in preclinical models. In CALR-mutant MPN, the path from discovery to mechanistic understanding to direct therapeutic targeting has advanced rapidly. The longer-term goal remains clonally-selective therapies that modify the disease course in patients.

Selective targeting of mutated calreticulin by the monoclonal antibody INCA033989 inhibits oncogenic function of MPN

AbstractMutations in calreticulin (mutCALR) are the second most common drivers of myeloproliferative neoplasms (MPNs) and yet, the current therapeutic landscape lacks a selective agent for mutCALR-expressing MPNs. Here, we show that the monoclonal antibody INCA033989 selectively targets mutCALR-positive cells. INCA033989 antagonized mutCALR-driven signaling and proliferation in engineered cell lines and primary CD34+ cells from patients with MPN. No antibody binding or functional activity was observed in the cells lacking mutCALR. In a mouse model of mutCALR-driven MPN, treatment with an INCA033989 mouse surrogate antibody effectively prevented the development of thrombocytosis and accumulation of megakaryocytes in the bone marrow. INCA033989 reduced the pathogenic self-renewal of mutCALR-positive disease-initiating cells in both primary and secondary transplantations, illustrating its disease-modifying potential. In summary, we describe a novel mutCALR-targeted therapy for MPNs, a monoclonal antibody that selectively inhibits the oncogenic function of MPN cells without interfering with normal hematopoiesis.

Real-world status of treatment for lymphoid neoplasms developed during the course of myeloproliferative neoplasms in Japan

ABSTRACT Objectives: Patients with myeloproliferative neoplasms (MPNs) are at higher risk of developing secondary malignancies. In this study, we focused on patients with MPNs that complicated lymphoid neoplasms. To analyze the real-world status of lymphoid neoplasm treatment in patients with pre-existing MPNs in Japan, we conducted a multicenter retrospective study. Methods: Questionnaires were sent to collect the data on patients who were first diagnosed with either polycythemia vera, essential thrombocythemia or myelofibrosis and who later were complicated with lymphoid neoplasms defined as malignant lymphoma, multiple myeloma, or chronic lymphocytic leukemia/small cell lymphoma. Results: Twenty-four patients with MPNs complicated by lymphoid neoplasms were enrolled (polycythemia vera, n = 8; essential thrombocythemia, n = 14; and primary myelofibrosis, n = 2). Among these, diffuse large B-cell lymphoma (DLBCL) was the most frequently observed (n = 13, 54.1%). Twelve (92.3%) of the patients with DLBCL received conventional chemotherapy. Among these 12 patients, regarding cytoreductive therapy for MPNs, 8 patients stopped treatment, one continued treatment, and two received a reduced dose. Consequently, most patients were able to receive conventional chemotherapy for DLBCL with a slightly higher dose of granulocyte colony-stimulating factor support than usual without worse outcomes. All 3 patients with multiple myeloma received a standard dose of chemotherapy. Conclusion: Our data indicate that if aggressive lymphoid neoplasms develop during the course of treatment in patients with MPNs, it is acceptable to prioritize chemotherapy for lymphoma.

Abstract TP224: Timing of Ischemic and Hemorrhagic Stroke With Myeloproliferative Neoplasm Diagnosis Among Veterans

Myeloproliferative Neoplasms (MPNs) are rare acquired stem cell disorders, consisting of Essential Thrombocythemia (ET), Polycythemia Vera (PV), and Primary Myelofibrosis (PMF), complicated by thrombohemorrhagic events such as stroke. The timing of ischemic stroke (IS) and hemorrhagic stroke (HS) in relation to MPN diagnosis among Veterans remains undefined. We utilized the Veterans Affairs Informatics and Computing Infrastructure database from 1/1/2006 - 1/26/2023 and included 586,555 Veterans from Illinois, the state most representative of the US population. ICD-9 and -10 codes identified Veterans with stroke and MPN. Fisher’s exact test was utilized to compare MPNs vs. non-MPNs. There were 237 MPNs and 15,221 non-MPNs with IS while there were 26 MPNs and 1,567 non-MPNs with HS (Table 1). There were no differences in age of stroke diagnosis among MPNs vs. non-MPNs. There were higher rates of hypertension, smoking, and heart failure among MPNs vs. non-MPNs with IS. Majority of IS was diagnosed either more than 3 months before MPN diagnosis (N=115, 48.5%) or more than 5 years after diagnosis (N=98, 41.4%). Among MPNs with HS, stroke was predominantly diagnosed (N=14, 53.8%) more than 3 months before and (N=7, 26.9%) within 3 months of MPN diagnosis (Figure 1). Among MPNs there was recurrence of stroke among 45.18% of IS and 40% of HS. Although MPN are infrequent causes of stroke, these findings suggest continued surveillance for MPN and workup for driver mutations after a stroke diagnosis. This study also suggests consideration for dual antiplatelet therapy for MPNs with cardiovascular risk factors to prevent IS and a multidisciplinary approach between neurology and hematology.

Outcomes of Patients with Accelerated/Blast-Phase MPNs That Received Allogeneic Stem-Cell Transplant in the Current Era of Myeloid Therapies

Background Patients (pts) with Philadelphia chromosome-negative (Ph-neg) myeloproliferative neoplasms (MPNs) that progress to an accelerated phase (AP) or blast phase (BP) have a median overall survival (mOS) of less than 1 year (Odenike, Blood 2018; Tam et al, JCO 2009). Allogeneic hematopoietic stem cell transplant (allo-HCT) is the only curative approach for MPN-AP/BP, but mOS remains about a year for pts with MPN-BP that undergo allo-HCT (2005-2019) (Orti et al, AJH 2023). We previously reported on outcomes of pts with MPN-AP/BP treated in the current era of myeloid therapies (2017 onward) and found a mOS of 0.73 years in a multi-center cohort of 80 pts (Patel et al, ASH 2022). We aim to characterize treatment patterns and outcomes of pts with MPN-AP/BP that underwent allo-HCT via a multi-center retrospective analysis. Methods Retrospective chart review was performed at 8 institutions to identify pts with pathology-confirmed Ph-neg MPN-AP/BP diagnosed in 2017 or later. All patients that met inclusion criteria were included. Response was assessed using 2017 European Leukemia Net AML (2017 ELN) criteria (Dohner et al, Blood 2017). OS from diagnosis of MPN-AP/BP and from time of allo-HCT was calculated utilizing Kaplan-Meier analysis. Results The full MPN-AP/BP cohort consists of 180 pts. This analysis includes 58 pts that underwent allo-HCT for MPN-AP/BP across 8 institutions. Median age at MPN-AP/BP diagnosiswas 64 years. The most common therapies for chronic-phase MPN included hydroxyurea (55%), JAK inhibitor (JAKi) (26%), interferon (9%), and hypomethylating agent (HMA, 4%). No patient previously received allo-HCT for chronic-phase MPN. Driver mutations included JAK2 (60%), CALR (22%), MPL (10%), but some pts had triple-negative disease (7%). Sixty-two percent of pts had high-risk disease by 2017 ELN criteria. Forty-four pts had next-generation sequencing performed at time of MPN-AP/BP diagnosis. The most frequently co-occurring mutations detected at time of MPN-AP/BP diagnosis were ASXL1 (30%), SRSF2 (23%), RUNX1 (23%), TP53 (18%), TET2 (16%), IDH2 (11%), U2AF1 (7%), EZH2 (7%), and IDH1 (9%). Additional characteristics are in Table 1. One pt proceeded directly to allo-HCT at time of MPN-AP/BP diagnosis. Frontline therapies (1L) for MPN-AP included intensive chemotherapy (IC) (n=26), hypomethylating agent (HMA) + venetoclax (VEN) (n=16), HMA monotherapy (n=14), and low-dose cytarabine (LDAC) + VEN + JAKi (n=1). Responses are summarized in Table 2A. Thirty-seven pts proceeded to allo-HCT after 1L therapy (17 after IC, 10 after HMA+VEN, 9 after HMA, 1 after LDAC+VEN+JAKi). Twenty pts proceeded to allo-HCT after 2 nd-line and beyond (2L+) therapies. Therapies utilized and responses are summarized in Table 2B. Transplant-specific characteristics include 15 pts receiving myeloablative conditioning while 43 received reduced-intensity conditioning. Donor source was matched unrelated donor for 33 pts, haploidentical donor for 10 pts, matched related donor for 9 patients, mismatched unrelated donor for 5 patients, and cord blood for 1 patient. Twenty-two pts developed acute GVHD while 15 developed chronic GVHD. Amongst the 58 pts that underwent allo-HCT, 19 had relapse of disease and 8 patients received treatment after relapse. Amongst the full cohort of 180 patients, mOS was 0.72 years from time of MPN-AP/BP diagnosis. The mOS for the 58 pts that underwent allo-HCT was 1.82 years from time of MPN-AP/BP diagnosis and 1.30 years from time of allo-HCT. Survival analysis by initial MPN-AP/BP therapy demonstrated a mOS from time of MPN-AP/BP diagnosis of 1.9 years for HMA-based therapy, 1.9 years for HMA + VEN, and 1.5 years for IC ( p=0.006). We also analyzed mOS by disease status prior to transplant. Thirty-four pts had a complete response (CR) or CR with incomplete count recovery (CRi) with mOS of 1.7 years from time of allo-HCT, 12 pts with a partial response (PR) or morphologic leukemia-free state (MLFS) had mOS of 1.8 years, and 11 pts with treatment failure (TF) had mOS of 0.47 years (p=0.82). Conclusions Our cohort of 180 pts with MPN-AP/BP treated in the current era of myeloid therapies had limited survival with a mOS of 0.72 years from MPN-AP/BP diagnosis. Amongst the 58 pts that underwent allo-HCT the mOS was 1.82 years with longer mOS seen in pts that received HMA or HMA + VEN as their 1L therapy when compared to IC. This underscores the need for novel management strategies even in pts eligible for allo-HCT.

Cladribine-Based Chemotherapy for Patients with Secondary Acute Myeloid Leukemia (AML) Arising after Myeloproliferative Neoplasms (MPNs)

Introduction: Patients with acute myeloid leukemia (AML) evolving from myeloproliferative neoplasms (MPN) have poor survival with median of 4-6 months and limited therapeutic options that could induce up to 60% of overall responses, but they are usually transient. Cladribine added to a backbone regimen of high dose cytarabine [≥ 1 gram per m2 intravenously (IV) for up to 5 days] and idarubicin (CLIA; further HD-clad) or low dose cytarabine [LDAC; 20 mg twice daily subcutaneously (SQ) for up to 10 days] alternating every two cycles with hypomethylating agent (HMA; regimen referred as LD-clad) have been routinely used in the treatment of AML at our institution. Objectives: We sought to investigate the efficacy and outcome of using HD-clad or LD-clad in patients with AML arising from MPN at our center. Methods: This is a retrospective chart review of patients with AML after previous MPN who were treated with clad-based regimen at our center and had comprehensive clinical data, including next generation molecular sequencing at AML diagnosis (≥ 28-gene myeloid panel). Since HMA-based therapy is often utilized early in MPN patients with accelerated (blasts up to 20%) or blast phase (blasts ≥20 %), and since HMA-treatment can affect outcomes, we separately evaluated patient's outcomes based on their prior exposure to HMA. Responses were according to the 2007 ELN criteria for AML and overall survival was measured from therapy initiation for AML (Kaplan-Meier method with log-rank test). Results: The cohort includes 50 patients (median age 68 years, range 28-83; 66% males); 72%, 18%, and 10% received clad-based therapy as induction (frontline, FL), salvage 1 and salvage 2+, respectively. Among the 36 FL patients, 23 (64%) received LD-clad and 13 (36%) HD-clad regimen. 30% and 38% of patients treated with FL LD-clad and FL HD-clad were previously exposed to HMA therapy; the remaining patients were HMA-naïve. All patients in the salvage (S) setting were pre-treated with HMA. Patients' characteristics at the time of AML therapy initiation are depicted in Table 1. Median time between the diagnosis of MPN and clad-based AML therapy was 62 months (range, 1-299) for FL and 137 months (range, 26-263) for S settings. 64% of patients on FL therapy had advanced grade of reticulin fibrosis, 44% had complex cytogenetics and 56% had JAK2 mutation. Additional co-mutations were detected in 78% of FL patients, the most common (in descending order) being ASXL1, RAS, RUNX1 and TP53. Patients treated with FL HD-clad were significantly younger (median age of 61 vs 72 years for patients on FL LD-clad, p = 0.03, respectively). LD-clad was more common as FL therapy, and was used in 67% of HMA-naïve and 58% of HMA-exposed patients. The complete remission rate with and without count recovery (CR & CRi) for FL HD-clad was 62%: 75% in HMA naïve and 40% in HMA-exposed patients. CR/CRi for FL LD-clad was 43%; 50% in HMA naïve and 29% in HMA-exposed patients. 75% and 50% of patients without previous HMA-exposure were able to proceed with allogeneic stem cell transplantation (SCT) after FL HD-clad and FL LD-clad, respectively. The median OS of 30 months (95% CI, 7-unreached) was the longest in HMA-naïve patients treated with FL HD-clad regimen (Figure; median OS censored for SCT was 15 months, range: not estimated - 24). The median OS for salvage pts treated with LD-clad and HD-clad were 3.25 and 1.85 months, respectively. Among 38 patients without SCT, 10 (26%) lived for more than 12 months since therapy initiation, including 2 patients in the salvage setting. Conclusion: Cladribine added to chemotherapy is an effective approach in patients with AML from MPN, either as an induction therapy for those eligible for SCT or acceptable long-term therapy for non-eligible patients. Noticeably, almost 40% of patients without SCT were alive past 12 months since the initiation of LD-clad therapy. HMA pretreated patients had lower response rates and poor survival even in the frontline setting.

GLS in Philadelphia-negative and JAK2 V617F-driven myeloproliferative neoplasms (MPNs).

e15092 Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies. Nearly all MPNs are driven by somatic mutations in either: JAK2, CALR, or MPL. Although all three MPN driver mutations lead to constitutive activation of JAK/STAT signaling, JAK inhibitors are not curative and fail to alter disease progression and display unwanted side effects. Allogeneic stem cell transplantation remains the only curative therapy for MPNs, but is associated with substantial morbidity and mortality. Recently, glutaminolysis has been shown to play a critical role in cancer cell metabolism. During glutaminolysis, glutamine is metabolized through in a two-step reaction, the first of which involves the enzyme glutaminase (GLS) catalyzing the hydrolysis of glutamine to glutamate. Glutamate can then fuel for energy production into the TCA cycle, among several other possible metabolic fates. As many cancers have proven to be dependent on this pathway, targeting GLS has become an attractive therapeutic avenue. Because glutaminolysis has been understudied in MPNs, we sought to determine whether this pathway represents a novel, targetable vulnerability in MPNs. Methods: We evaluated the mRNA levels of GLS in peripheral blood mononuclear cells from 30 MPN patients and 5 healthy donors. We tested by qPCR for GLS and the protein levels of GLS by western blot in TF-1 cells, a human myeloid cell line, stably overexpressing either JAK2, MPL, or CALR mutated proteins. The latter also under treatment with the JAK inhibitor ruxolinitinib. We also evaluated the GLS enzyme activity through the use of a fluorimetric assay. Lastly, we tested the sensitivity of MPN cells to GLS inhibition with a GLS inhibitor, CB-839, which is currently in advanced phase clinical trials for other cancers including Myelodysplastic Syndromes. Results: GLS mRNA expression was increased in all MPN patients regardless of their driver mutation, where expression in JAK2V617F patients was higher in MF versus ET patients (p < 0.001). GLS protein expression and activity were increased in TF-1 cells expressing JAK2, MPL, and CALR mutations. We also found that GLS mRNA and protein expression was up-regulated in a JAK/STAT-dependent manner. Interestingly, despite increased expression of GLS across all MPN driver mutations, only JAK2 V617F cells demonstrated significant sensitivity to GLS inhibition with CB-839 in vitro and with preliminary data in vivo. We found that combination treatment with JAK inhibitor ruxolitinib further inhibited cell viability. Conclusions: GLS up-regulation is a common feature of all MPNs, and is JAK/STAT dependent. JAK2 V617F expressing cells show significant sensitivity to GLS inhibition. Combination treatment with ruxolitinib further enhances this effect. Treatment with CB-839 may thus represent a novel therapeutic avenue for JAK2 V617F+ MPNs.

Development of an enzyme-coupled activity assay for Janus kinase 2 inhibitor screening.

JAK2 transmits signals of several important cytokines, such as growth hormone and erythropoietin. The interest toward the therapeutic targeting of JAK2 was boosted in 2005, when the somatic JAK2 V617F mutation, responsible for the majority of myeloproliferative neoplasms (MPNs) was discovered. JAK2 inhibitors have been approved for MPN therapy and they are effective in alleviating symptoms and improving the quality of life of the patients, but they do not lead to molecular remission. This calls for the discovery of new compounds for JAK2-targeted therapeutic approaches. Here we describe the development of a fluorescence-based activity assay for the screening of versatile inhibitor types against JAK2. The assay was utilized to screen a diverse set of small molecule weight natural products and the assay performance was compared to that of differential scanning fluorimetry. We identified 37 hits and further analysis of the most potent hits revealed that most of them displayed non-ATP competitive binding modes. The hits were profiled against other JAK family members and showed distinctive selectivity profiles. The developed assay is consistent, simple and inexpensive to use, and can be utilized for inhibitor screening of diverse compound classes against all JAK family members.

Clonal Hematopoiesis in Myeloproliferative Neoplasms Confers a Predisposition to both Thrombosis and Cancer.

This review focuses on vascular complications associated with chronic myeloproliferative neoplasms (MPN) and more specifically aims to discuss the clinical and biological evidence supporting the existence of a link between clonal hematopoiesis, cardiovascular events (CVE), and solid cancer (SC). The MPN natural history is driven by uncontrolled clonal myeloproliferation sustained by acquired somatic mutations in driver (JAK2, CALR, and MPL) and non-driver genes, involving epigenetic (e.g., TET2, DNMT3A) regulators, chromatin regulator genes (e.g., ASXL1, EZH2), and splicing machinery genes (e.g., SF3B1). The genomic alterations and additional thrombosis acquired risk factors are determinants for CVE. There is evidence that clonal hematopoiesis can elicit a chronic and systemic inflammation status that acts as driving force for the development of thrombosis, MPN evolution, and second cancer (SC). This notion may explain the mechanism that links arterial thrombosis in MPN patients and subsequent solid tumors. In the last decade, clonal hematopoiesis of indeterminate potential (CHIP) has been detected in the general population particularly in the elderly and initially found in myocardial infarction and stroke, rising the hypothesis that the inflammatory status CHIP-associated could confer predisposition to both cardiovascular diseases and cancer. In summary, clonal hematopoiesis in MPN and CHIP confer a predisposition to cardiovascular events and cancer through chronic and systemic inflammation. This acquisition could open new avenues for antithrombotic therapy both in MPNs and in general population by targeting both clonal hematopoiesis and inflammation.

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Targeted Therapy for MPNs: Going Beyond JAK Inhibitors.

JAK inhibition is an effective means of controlling symptom burden and improving splenomegaly in patients with myeloproliferative neoplasms (MPNs). However, a majority of patients treated with JAK inhibition will have disease progression with long-term use. In In this review, we focus on the investigation of novel targeted agents beyond JAK inhibitors both in the chronic phase of disease and in the accelerated/blast phase of disease. Relevant targeted therapies in MPNs include BET inhibitors, BCL inhibitors, LSD1 inhibitors, PI3K inhibitors, IDH inhibitors, telomerase inhibitors, and MDM2 inhibitor. Agents within these classes have been investigated either as monotherapy or in combination with a JAK inhibitor. We summarize the prospective data for these agents along with detailing the ongoing phase III trials incorporating these agents. While JAK inhibition has been a mainstay of therapy in MPNs, a majority of patients will have disease of progression. JAK inhibitors also have limited anti-clonal effect and do not impact the rate of progression to the blast phase of disease. The novel therapies detailed in this review not only show promise in ameliorating the symptom burden of MPNs but may be able to alter the natural history of disease.

Interferon and myeloproliferative neoplasms: Evolving therapeutic approaches.

Interferons (IFNs) are a diverse group of cytokines whose potent antitumor effects have piqued the interest of scientists for decades. Some of the most sustained clinical accomplishments have been in the field of myeloproliferative neoplasms (MPNs). Here, we discuss how both historical and novel breakthroughs in our understanding of IFN function may lead to more effective therapies for MPNs. The particular relevance and importance of modulating the novel IFN-regulated ULK1 pathway to optimize IFN responses is highlighted.

Escape from Type II JAK2 Inhibition in MPN Depends on AXL/MAPK Activation and Is Targetable

Myeloproliferative neoplasms (MPN) are characterized by dysregulated JAK2 signaling. Clinical type I JAK2 inhibitors as ruxolitinib are hampered by limited disease-modifying effects and occurrence of resistance. Based on pre-clinical data, novel type II JAK2 inhibitors as CHZ868 effectively stabilize inactive JAK2, reduce MPN clones and overcome resistance to ruxolitinib. We study whether MPN cells could escape from type ll JAK2 inhibition, and characterize therapeutic approaches. To assess potential escape from type II JAK inhibition, JAK2V617F MPN cells incl. SET2, UKE-1 and Ba/F3 EPOR Jak2V617F were exposed long-term to CHZ868. Outgrowing cells showed significantly increased IC50 to CHZ868 and reduced susceptibility for apoptosis induction. No second-site mutations in JAK2 were detected. Phosphoproteomic analysis identified >2500 differentially phosphorylated sites with most prominent activation of the MAPK pathway in JAK inhibitor resistant cells. Type II JAK inhibition persistently suppressed JAK2 - STAT3/5 signaling, while MAPK target activation incl. MEK and ERK was prominent also by phospho-immunoblot suggesting MAPK pathway signaling as a driver of escape from type II JAK inhibition. To characterize the molecular basis, we profiled differential gene expression and chromatin accessibility by RNA-sequencing and ATAC-sequencing in MPN cells escaping JAK inhibition. Paired analysis of RNA-seq/ATAC-seq revealed enrichment of H3K27me3 and PRC2 signatures with overall more compacted chromatin. Expression of AXL tyrosine kinase known to signal through the MAPK pathway, was significantly increased along with enriched RAS signaling target gene patterns by GSEA. Motif analysis suggested differential transcriptional programs with loss of ETS and increased accessibility of GATA transcription factor family binding sites. Enhanced AXL expression was confirmed by immunoblotting and depletion of AXL by AXL-specific shRNA knock-down resensitized resistant cells to CHZ868 with decreased IC50. Since these findings pointed towards a role of AXL activation for escape of MPN cells from type II JAK inhibition, we targeted AXL activation using respective inhibitors. AXL inhibitor bemcentinib and AXL/FLT3 inhibitor gilteritinib reduced proliferation of resistant cells with up to 5.6-fold and 3.4-fold decreased IC50, respectively. Given that type II JAK inhibition with CHZ868 maintained suppression of JAK2-STAT3/5 activation in resistant cells, we combined JAK2 and AXL inhibition. Notably, dual JAK2 / AXL inhibition with CHZ868 / bemcentinib or CHZ868 / gilteritinib showed most pronounced inhibitory effects with IC50 similar to values seen in JAK inhibitor sensitive cells and effective induction of apoptosis. Resistant MPN cells engrafted subcutaneously into immunocompromised NSG mice and resulted in robust tumor growth despite JAK2 inhibitor treatment. However, AXL inhibition with bemcentinib reduced progression of type II JAK inhibitor resistant tumors. Combined JAK2 and AXL inhibition showed most pronounced suppression of subcutaneous tumor growth with minimal progression. Given that inhibitors of MAPK signaling targeting e.g. MEK-ERK activation, are in clinical use for other malignancies with favorable tolerability profiles, we then characterized combined JAK2 / MAPK inhibition to overcome AXL-MAPK driven escape of MPN cells from type II JAK inhibitors. Combination of CHZ868 and MEK inhibitor trametinib significantly reduced subcutaneous tumor growth in NSG mice similarly to JAK2 / AXL inhibition. Expression of MAPK pathway effectors as DUSP6 was substantially reduced. Furthermore, intravenous engraftment of JAK inhibitor resistant cells into NSG mice led to substantial bone marrow (BM) infiltration shown by luciferase imaging and hCD45 immunohistochemistry despite treatment with CHZ868. Combined JAK2 / MAPK inhibition with CHZ868 and trametinib significantly reduced BM infiltration by luciferase imaging and histology supporting that escape from type II JAK inhibition is dependent on AXL-MAPK activation. In summary, we report on a novel mechanism of AXL-MAPK driven escape from type II JAK2 inhibition which is targetable in vitro and in vivo. Our findings highlight that rationally designed combination therapy approaches can restore sensitivity to JAK inhibitor therapy in MPN, which may have potential implications for other malignant settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative MRI Identifies Heterogeneous Bone Marrow Treatment Responses in a Mouse Model of Myelofibrosis

Myeloproliferative neoplasms (MPNs), a class of blood cancers, typically arise from mutations in the thrombopoietin receptor (MPL), JAK2, or calreticulin that constitutively activate JAK/STAT signaling in hematopoietic stem and progenitor cells. Myelofibrosis (MF), the most aggressive of the MPNs, can arise as a primary neoplasm or secondary to other MPNs. MF is characterized by progressive fibrosis of bone marrow (BM), destruction of the hematopoietic niche, and eventual BM failure. Recently, the focus of treatment in MF has shifted toward restoration of healthy BM architecture and a functional hematopoietic niche rather than focusing solely on alleviating symptoms. Accomplishing this paradigm shift in drug development and therapy requires new approaches to monitor spatial and temporal changes in BM in patients during treatment. We present a quantitative magnetic resonance imaging (MRI) approach to analyze key pathologic changes in BM in a mouse model of MPN driven by MPLW515L. To evaluate disease extent, progression, and response to therapy in mice, we use anatomical MRI for spleen volume and three quantitative MRI metrics for BM: 1) apparent diffusion coefficient (ADC), 2) proton density fat fraction (PDFF), and 3) magnetization transfer ratio (MTR). PDFF and ADC assess the fat content and water movement of the BM and are useful in identifying cellularity changes such as hypercellularity in MPNs and MF. MTR probes changes in macromolecular structure, such as those observed with increasing fibrosis in MF. We used quantitative MRI to measure response to treatment in BM and change in spleen volume in response to three drugs: ruxolitinib (JAK1/2 inhibitor), fedratinib (JAK2 inhibitor), and navitoclax (BCL-2/BCL-xL inhibitor). Within 10 days after starting treatment, diseased spleen volumes in the ruxolitinib group (~3.5x healthy volume) had the greatest average reduction in spleen volume measured by MRI (-73.5% ± 11.4%) followed by the fedratinib group (-52.7% ± 16.7%). Interestingly, the spleens of every ruxolitinib-treated mouse displayed consistent and prolonged spleen volume reductions, while only a subset of mice in the other treatment groups (~70% fedratinib mice; ~33% navitoclax mice) demonstrated reductions in spleen volume. Despite the robust spleen response in ruxolitinib-treated mice, only some of the mice (~60%) exhibited sustained changes in BM ADC, MTR, and PDFF signals expected with reversion to healthy BM (decreased ADC, decreased MTR, and increased PDFF). In mice treated with fedratinib or navitoclax, even fewer mice (~40% and ~33% respectively) showed reversion toward healthy BM. Importantly, we observed spatial heterogeneity of BM response within the tibia of single mice, where proximal and distal tibia regions often responded disparately (~49% of cases). Because improving BM cellularity and fibrosis is increasingly the target of therapy in MPNs and in MF specifically, there is a distinct need for better techniques for longitudinally evaluating variations in treatment efficacy in living subjects. In this study, we demonstrate that the combination of three quantitative MRI metrics (ADC, PDFF, and MTR) can detect the impact of therapy on the BM in a preclinical mouse model of MPN/MF. Interestingly, we identified pronounced heterogeneity at multiple levels during treatment: 1) differences in changes in spleen volume versus BM; 2) disparate treatment responses of mice within a treatment group; and 3) contrasting regional treatment responses of the BM within a single mouse. Because these quantitative MRI metrics are directly translatable to clinical medicine and can identify variations in treatment efficacy in BM, both between and within subjects, this study sets the stage for future drug development and analysis of therapeutic interventions on the BM of patients with MF.

Molecularly Targeted Combination Therapy for Advanced Phase Myeloproliferative Neoplasm: MPN-RC 119

Background: Patients with myeloproliferative neoplasms (MPNs) that develop accelerated (AP) or blast phase (BP) disease have poor outcomes and limited treatment options. The median overall survival (OS) for patients with MPN-BP is 3-5 months (Mesa, Blood 2005; Mascarenhas, CLML 2016), and 12 months for patients with MPN-AP. Myelofibrosis (MF) patients with 5-9% blasts in the peripheral blood (PB) have a similar outcome as patients with MPN-AP (Massarova, CLML 2021). IDH mutations occur in 20-25% of patients with MPN-BP and the acquisition of these mutations is associated with both an increased risk of leukemic transformation and reduced OS (Dunbar, blood 2020). IDH2R140Q-Jak2V617F mice treated with a combination of JAK1/2 inhibitor (ruxolitinib) and an IDH2 inhibitor (enasidenib) showed complete resolution of splenomegaly, normalization of blood counts, and blast resolution (McKenney, JCI 2018). Here we report the preliminary results of the first molecularly targeted therapy for MPN AP/BP patients harboring IDH2 mutations utilizing combination treatment with ruxolitinib and enasidenib. Methods: A phase 2, multicenter trial of combined ruxolitinib and enasidenib is being conducted in patients with MPN-AP/BP or MF in chronic phase (CP) with 4-9% PB blasts and an IDH2 Mutation (NCT04281498). MPN-AP/BP and MF-CP Patients receive enasidenib 100mg and 50mg daily respectively, with dose increases after cycle 1 for MF-CP. Initial ruxolitinib doses were determined based on platelet counts at enrollment for patients without previous ruxolitinib treatment or continuation of current treatment dose. Response assessment occurred every 3 cycles for MPN-AP/BP using modified Cheson criteria and every 6 cycles for MF-CP using IWG/ELN criteria. Results: Six patients have been enrolled (5 MPN-AP/BP and 1 MF-CP). 50% were male with a median age of 74 years (range 58-81). (Table 1). Patients were treated for a median of 5.5 cycles (1-13) with a median follow up of 26 weeks (2.7-53). At the time of reporting, two patients remain on trial. Four discontinued treatment; disease progression (n=1), physician discretion (n=1, after no response observed after 3 cycles), allogeneic stem cell transplant (n=1), withdrew consent (n=1). Grade 3/4 adverse events (AEs) were reported in five of the six (83%) patients. All grade 4 AEs were hematological events, as well as the majority of the grade 3 AEs. One patient (16.7%) had grade 3 liver function test abnormalities, one patient (16.7%) had grade 3 hyperbilirubinemia and one patient (16.7%) had grade 3 neutropenic fever. QT prolongation and evidence of a differentiation syndrome were not observed. AEs are summarized in Table 2. Of the five MPN-AP/BP patients, one pt was not evaluable for response (removed after 1 cycle due to disease progression). The ORR was 50% in the evaluable cohort and 40% in the intention to treat analysis. Two patients achieved complete remission (CR). One patient achieved CRi after 3 cycles and has maintained the response after 13 cycles, one patient achieved CRi after 3 cycles, and CR after 4 cycles, and has maintained the response after 9 cycles. Two patients had stable disease after 3 and 6 cycles. Conclusion: Combination treatment with ruxolitinib and enasidenib appear safe with manageable toxicity profile. The majority of grade 3/4 AEs were hematologic as expected in this advanced patient population. Results are encouraging and warrant continued evaluation of this IDH2 mutant defined poor prognostic group. Trial enrollment is ongoing and mature results will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-053 Drug-Induced Hypertension in Classical Philadelphia-Negative Myeloproliferative Neoplasms (MPNs): Systematic Review

Secondary hypertension, e.g., drug-induced hypertension (DI-HTN), can occur in patients with solid/hematological cancers. However, the relationship between anticancer therapy and DI-HTN in classical Philadelphia-negative MPNs is unclear. Investigate this research question via a systematic review. Comprehensive search in PubMed/MEDLINE, SCOPUS and Web of Science. Publications that reported DI-HTN as an adverse effect (AE) of anticancer therapy for MPNs were included in the qualitative/quantitative synthesis. Search strategy identified 590 records. After removal of duplicates/screening of titles/abstracts, 87 papers were selected for full-text review. After inclusion/exclusion criteria were applied, 12 manuscripts entered into the qualitative/quantitative synthesis. Ruxolitinib's effect (RUX) on systolic (SBP)/diastolic (DBP) blood pressure is controversial: one study reported no effect on SBP/DBP, whereas in another SBP but not DBP increased. In RESPONSE-2 trial, DI-HTN developed as non-hematological AE [grade 3-4 (6%, n=5); grade 1-2 (3%, n=2)] in 9% (n=7) of 74 polycythemia vera (PV) patients enrolled. RESPONSE-2 secondary analyses reported RUX-induced hypertension as the most frequent grade 3/4 AE in the RUX/RUX cross-over arms. Interferon did not cause DI-HTN in RESPONSE and RESPONSE-2 trials. RUX+decitabine in accelerated/blast phase MPNs (n=25) caused 3 DI-HTN cases: grade 1/2 (n=2), grade 3+ AE (n=1). RUX+decitabine resulted in 2 DI-HTN cases (grade 3 AE in 21 MPNs in accelerated/blast phase). RUX+danazol in 14 primary/secondary myelofibrosis (PMF/SMF) subjects lead to 1 DI-HTN case (n=1, 7.1%) in a multicentric phase 2 study. Anagrelide caused DI-HTN as AE in 9 essential thrombocythemia (ET) subjects, however, hydroxyurea caused DI-HTN in 1 of 146 patients. Italian ET registry retrospective evaluation reported DI-HTN in 3.5% (n=8) of 232 anagrelide-treated subjects. DI-HTN occurred as dose-limiting toxicity in an open-label, dose-escalation assessment of AT9283 (Aurora kinases inhibitor) in advanced PMF/SMF and refractory AML. Givinostat caused DI-HTN (grade 3 AE) in 1 of 50 PV subjects. RUX (alone/in combination with decitabine/danazol), anagrelide, AT9283, and givinostat can cause DI-HTN in classical Philadelphia-negative MPNs.

Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs

AbstractCalreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

Management of Myeloproliferative Neoplasms (MPNs)

To provide up to date guidance, practice recommendations and highlight barriers to medication adherence in the long-term management of chronic myeloproliferative neoplasms (MPNs). Current drug therapy for MPN is not curative and has not been shown to prolong survival. The main indication for treatment is the prevention of thrombosis and medication adherence remains a challenge in this group of patients. Identifying potentially modifiable barriers to medication adherence including primary nonadherence and non-persistent adherence enables timely interventions to be put in place and improve overall medication adherence. A systematic review of peer-reviewed literature and expert opinions was performed using electronic databases (PubMed, EMBASE, MEDLINE, and Web of Science) that were searched for articles reporting MPN and medication adherence. Discussions A case vignette is discussed throughout the article and expert opinion with international peer reviewed guidelines that are authored to support clinical decision making at the point of care were utilised. The evidence base was combined with more practical/clinical (data based) insight from real world clinical practice. Adoption of a broad range of digital health care activities and services in the health care system (telehealth applications) by the advanced practice providers (Non-Medical Prescribers-NMPs) in MPN clinics included medication prescribing and management, oral drug compliance and adherence evaluations, interventions, chronic care management, counselling and patient education on treatments. Current drug therapy for MPN is neither curative nor has it been shown to prolong survival, and medication adherence remains a challenge in this group of patients. The longevity of the patients' disease course may contribute to the high risk of non-adherence in this patient cohort. Poor adherence to long-term therapies severely compromises the effectiveness of treatment. Adherence interventions should be tailored to the needs of the patient in order to achieve maximum impact. Interventions aimed at improving adherence provide the best experience and outcome for the patient and their families and can have a profound impact on the quality of life and mitigation of disease consequences.

JAK2 in Myeloproliferative Neoplasms: Still a Protagonist.

The discovery of the activating V617F mutation in Janus kinase 2 (JAK2) has been decisive for the understanding of myeloproliferative neoplasms (MPN). Activated JAK2 signaling by JAK2, CALR, and MPL mutations has become a focus for the development of targeted therapies for patients with MPN. JAK2 inhibitors now represent a standard of clinical care for certain forms of MPN and offer important benefits for MPN patients. However, several key aspects remain unsolved regarding the targeted therapy of MPN with JAK2 inhibitors, such as reducing the MPN clone and how to avoid or overcome a loss of response. Here, we summarize the current knowledge on the structure and signaling of JAK2 as central elements of MPN pathogenesis and feature benefits and limitations of therapeutic JAK2 targeting in MPN.