Mouse Models Of Myeloproliferative Neoplasms Research Articles

Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Upregulation of Platelet Transcripts As Biomarkers of Fibrotic Progression in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of clonal hemopoietic disorders that have a proliferative phase (including essential thrombocythemia (ET) and polycythemia vera (PV)), and which can progress to advanced disease such as myelofibrosis (MF) or acute leukemia. This is thought to be due in part to progressively dysfunctional megakaryocytes driving reticulin deposition in the bone marrow. In previous work, we demonstrated that the platelet transcriptome in MPN differs from normal, and the difference is most marked with increased reticulin (Guo et al., 2020). A putative 3-gene signature ( CCND1, H2AFX and CEP55) could discriminate between patients with and without increased reticulin fibrosis. To progress this, we have profiled the transcriptome of platelets from a further 173 MPN patients were analyzed using next generation sequencing. CCND1, H2AFX and CEP55 were upregulated in platelets from patients with MF compared to ET (4.6-fold, padj<0.001, 9.0-fold, padj<0.001 and 10.5-fold, padj<0.001, respectively) and to PV (3.9-fold, padj<0.001, 9.2-fold, padj<0.001, 12.0-fold, padj<0.001). Application of the established 3-gene signature had a positive predictive value of 78%, a negative prediction value of 90% and accuracy of 88% for MF. To assess the temporal changes in these gene transcripts in platelets and the potential utility to predict fibrotic progression, we then performed longitudinal analyses in two MPN mouse models. Jak2V617F knock-in mice ( n=25) with stable serially transplantable disease, which resembles PV (Mullally et al., 2010), were sacrificed at 4, 8, 12, 16 and 20 weeks post-transplant, and controls ( n=5) at 20 weeks. Blood counts were performed, and spleen and sternum harvested and stained with hematoxylin and eosin, and for reticulin. By 20-weeks, the platelet and leucocyte counts and hemoglobin had increased as expected. There was splenomegaly with extramedullary hematopoiesis and a mild increase in marrow reticulin. There was no significant up-regulation of Ccnd1, H2afx or Cep55 in platelet transcripts at any timepoint compared to the control mice. This data showed stability of the mouse PV phenotype and platelet transcripts, in keeping with the chronic phase of MPN. Mice with MPLW515L ( n=8) (Pikman et al., 2006) were sacrificed at 3 and 6 weeks post-transplant, and control mice ( n=4) at 6 weeks. At 3-weeks post-transplant Cep55 (131.6-fold, padj<0.001) and H2afx (1.7-fold, padj<0.01) had increased in the MPLW515L mice and there was no increase in reticulin in the bone marrow. At 6-weeks platelet and leucocyte counts had increased, there was splenomegaly with extramedullary hematopoiesis including marked megakaryocytosis and marrow reticulin. On platelet transcript analysis Cep55 (154.6-fold, padj<0.001) and H2afx (2.4-fold, padj<0.001) were significantly increased; there was no significant change for Ccnd1. Upregulation of Cep55 and H2afx in the MPLW515L mouse occurred prior to the accumulation of bone marrow reticulin and development of MF phenotype. This suggests that up-regulation of Cep55 and H2afx in platelets may be a predictor of disease progression from ET and PV to MF. For MPN patients, a deviation from steady-state platelet transcript levels, specifically for H2AFX and CEP55, may infer a biological transition towards the fibrotic phenotype. This novel approach utilizing platelet transcript analysis may therefore have potential for serially monitoring MPN patients and the early identification of genomic changes that may herald disease transformation to MF.

SHP2 Inhibition Enhances JAK2 Inhibitor Therapy in Preclinical Models of Myeloproliferative Neoplasms

The classic myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are driven by aberrant activation of the JAK2 kinase, primarily by mutations in 3 different genes, JAK2, MPL, and CALR. JAK2 inhibitor therapy is generally effective at reducing MPN patient constitutional symptoms and thus can improve quality of life but has minimal effect on reducing mutant driver allele burden and does not readily alter the course of disease, reflecting the ability of MPN-driving cells to persist during therapy. Thus, the ability of current anti-JAK2 therapies to significantly modify underlying disease biology is limited. Long-term studies of the JAK2 inhibitor ruxolitinib in myelofibrosis patients have, however, identified survival benefits, yet this may be due to anti-inflammatory and spleen reducing impacts. Ultimately, the inability of ruxolitinib and other approved JAK2 inhibitors to effectively antagonize allele burden indicates alternative therapeutic approaches are needed to overcome the persistent survival of MPN-driving cells during JAK2 inhibitor therapy. Efforts to identify mechanisms that can thwart the effects of JAK2 inhibition in MPN cells have highlighted activation of the RAS-ERK pathway, including mechanisms of RAS-ERK signaling that are independent of JAK2. ERK or MEK inhibition have been shown to enhance the activity of ruxolitinib in MPN mouse models, but monotherapeutic inhibition of these kinases displayed limited efficacy. The SHP2 (Src homology region 2 domain-containing phosphatase-2) tyrosine phosphatase mediates RAS activation and subsequent MEK/ERK signaling downstream of cytokine and growth factor receptors. SHP2 plays a role in oncogenic signaling and in resistance to kinase targeted therapies, exemplified in both solid tumor and leukemia models. Given the evidence that activation of RAS-ERK signaling can antagonize the effects of JAK2 inhibition and the fact that SHP2 mediates RAS activation via multiple mechanisms downstream of numerous growth factor and cytokine receptors, we hypothesized that SHP2 may contribute to the survival of MPN cells during JAK2 inhibitor treatment and may provide a therapeutic target to improve current anti-JAK2 therapies. Using preclinical MPN models, we aimed to block RAS activation by inhibiting SHP2 to assess the potential SHP2 inhibition may have as a monotherapy or in combination with JAK2 inhibition. We found that the SHP2 inhibitors RMC-4550 and SHP099 enhanced growth inhibition of MPN model cell lines (e.g., SET2 and UKE1) in combination with ruxolitinib, effectively preventing ruxolitinib persistent growth. Concomitant treatment with both inhibitors led to enhanced inhibition of RAS-GTP levels and ERK activation and increased apoptosis compared to single agents. SHP2 inhibition antagonized the neoplastic growth of primary hematopoietic progenitor cells from MPN patients and synergized with ruxolitinib in all patient samples assessed. In an MPN mouse model driven by MPL-W515L, RMC-4550 antagonized MPN phenotypes, including leukocytosis, hepatosplenomegaly, and allele burden. RMC-4550 treatment diminished levels of a subset of pro-inflammatory cytokines, including IL-1β, CXCL9, and TNFα, among others. The combination of RMC-4550 with ruxolitinib not only suppressed advancing leukocytosis but led to white blood cell levels lower than at treatment initiation (Panel A) and suppressed thrombocytosis compared to ruxolitinib alone. The combination also led to enhanced inhibition of hepatomegaly, extramedullary hyperplasia, megakaryocyte hyperplasia, and allele burden, and a trend toward improved fibrosis. The SHP2 inhibitor RMC-4550 enhanced the survival of mice (following termination of treatment analyses) as both a monotherapy (p < 0.0001 compared to vehicle) and in combination with ruxolitinib (p < 0.01 compared to ruxolitinib monotherapy). Importantly, the combination of SHP2 inhibition using RMC-4550 with JAK2 inhibition using ruxolitinib for 4 weeks in wildtype mice was well tolerated with respect to hematologic parameters and exemplified by no effect on body weight (Panel B). Given SHP2 inhibitors are already undergoing clinical evaluation in patients with solid tumors, our findings suggest that SHP2 is a therapeutic target with potential to be rapidly translated to clinical assessment for MPN patients.

Bomedemstat (IMG-7289), an LSD1 Inhibitor, Manages the Signs and Symptoms of Essential Thrombocythemia (ET) While Reducing the Burden of Cells Homozygous for Driver Mutations

The ultimate goal in the treatment of ET is to eliminate mutant cells with the capacity to both proliferate and self-renew. Lysine-specific demethylase-1, LSD1, is an enzyme critical for regulating the proliferation of hematopoietic stem cells and the maturation of progenitors (Sprussel et al. 2012). Bomedemstat is an orally active LSD1 inhibitor that reduces platelets, inflammatory cytokines, and the mutant cell burden in mouse models of myeloproliferative neoplasms (Jutzi et. al., 2019). All current treatments - hydroxyurea, interferon, or anagrelide - have their limitations and do not substantially affect the natural history of ET. IMG-7289-CTP-201 was a global, open-label, Phase 2b study of bomedemstat (IMG-7289, MK-3543) taken once daily for 24+ weeks in patients with ET resistant to or intolerant of at least one standard treatment (NCT04254978). The primary endpoint was reduction of platelets to <400 x 10 9/L in the absence of new thromboembolic events. Exploratory endpoints included changes in frequencies of mutant alleles (VAF) assessed by deep sequencing (median exonic depth =1784 reads) of 261 genes of germline and somatic DNA. Homozygosity of mutant alleles was imputed when VAF >50% and/or loss-of-heterozygosity (LOH) was evident based on the difference between the minor allele frequency of flanking single nucleotide polymorphisms (“MAF method”) in germline versus granulocyteDNA. Single cell genotypes (scDNA) (Tapestri ®) were determined in stem/progenitor (CD34+) and monocytes (CD14+) cells in patients with LOH (using the MAF method) showing a decrease in LOH in whom follow-up samples were available. For patients treated ≥24 wks, 95% (61/64) had reduced platelet counts to ≤400 x 10 9/L without new thromboembolic events, in a median time of 10 wks. In the first 48 weeks, four patients experienced a thromboembolic event ( per PI) (1 PE, 2 TIAs, and 1 transient global amnesia). Of the 14/73 (19%) patients with Day 1 WBC count >10 x 10 9/L, 100% treated for ≥24 wks had reduced the WBC count to ≤10 x 10 9/L; mean Hb remained stable. In patients with baseline MPN-SAF TSS >20 (28/73), at Wk 24, 78% (18/23) experienced an improvement with 52% (12/23) improving ≥10 points. The median baseline score for worst fatigue was 4.0 (N=73); by Wk 24, the score had improved to 3.0 (N=62). Among all patients (N=73), the most common AEs regardless of causality were dysgeusia (55%), constipation (38%), thrombocytopenia (34%), and arthralgia (27%). Twenty-four patients reported serious AEs (SAE), with related events ( per PI) in 4 patients. Twenty-one patients discontinued treatment, 11 due to AEs (3 for dysgeusia), 7 subject's decisions, 1 PI decision, 1 disease progression, and 1 unrelated death due to aspiration pneumonia. There have been no safety signals or deaths related to drug in patients treated for up to 979 days. At Wk 24, 86% patients remained on study and 75% at Wk 48. At ~Wk 24, 39 of 46 (85%) of evaluable patients had a decrease in driver VAF (Fig. 1). The greatest changes in VAF occurred in patients with cells homozygous for a driver mutation (Fig. 1). Nine of ten patients with homozygous driver clones had follow-up data: by bulk sequencing of germline+granulocyte DNA, 7 showed a reduction in the proportion of homozygous cells. In all cases tested, scDNA of CD14+ (monocytes) and CD34+ (stem/progenitor) cells were qualitatively similar to results obtained with bulk granulocyte sequencing (Fig.2). By bulk sequencing, the proportion of homozygous cells dropped by a mean of 66% (-4% to -100% (-100% = homozygosity not detectable)). Decreases in LOH by bulk and sc sequencing during treatment with bomedemstat were identified in patients with homozygosity at JAK2, CALR, and MPL. This study demonstrates that treatment with bomedemstat results in a consistent hematologic response in the majority of patients with ET. Further, deep bulk sequencing of germline+granulocyte DNA reveals LOH ( i.e., homozygosity) events with high accuracy that enables determination of the distribution of homozygote, heterozygote and wild-type genotypes. Furthermore, this study shows that sequencing germline+granulocyte DNA provides a reliable qualitative view of the impact of treatment on the genotypic distribution among CD34+ cells as revealed by scDNA genotyping. Finally, bomedemstat showed activity reducing stem/progenitor cells homozygous for mutations in JAK2, CALR, and MPL.

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.

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression.

Myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed single-cell RNA sequencing on serial MPN and sAML patient stem and progenitor cells, identifying aberrantly increased expression of DUSP6 underlying disease transformation. Pharmacologic dual-specificity phosphatase (DUSP)6 targeting led to inhibition of S6 and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling while also reducing inflammatory cytokine production. DUSP6 perturbation further inhibited ribosomal S6 kinase (RSK)1, which we identified as a second indispensable candidate associated with poor clinical outcome. Ectopic expression of DUSP6 mediated JAK2-inhibitor resistance and exacerbated disease severity in patient-derived xenograft (PDX) models. Contrastingly, DUSP6 inhibition potently suppressed disease development across Jak2V617F and MPLW515L MPN mouse models and sAML PDXs without inducing toxicity in healthy controls. These findings underscore DUSP6 in driving disease transformation and highlight the DUSP6-RSK1 axis as a vulnerable, druggable pathway in myeloid malignancies.

Crizotinib Has Preclinical Efficacy in Philadelphia-Negative Myeloproliferative Neoplasms.

The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are characterized by JAK/STAT pathway activation. JAK inhibitors are approved for MPN treatment, but persistence has been observed, due to JAK/STAT reactivation. Using MPN patient samples, JAK2-mutated cell lines, and MPN mouse models, we examined both the efficacy and mechanism by which crizotinib, the ALK/MET/RON/ROS1 inhibitor approved for the treatment of non-small cell lung cancer, alters MPN cell proliferation and JAK/STAT activation. We found that crizotinib suppresses proliferation and activation of JAK/STAT signaling, and decreases the disease burden in the JAK2V617F mouse model of MPN. Furthermore, we found that crizotinib could overcome JAK inhibitor persistence to ruxolitinib. Interestingly, phosphorylation of the crizotinib target RON kinase was enhanced in ruxolitinib-persistent cells. We show that phospho-JAK2 and phospho-RON can physically interact to sustain JAK/STAT signaling, and that the combination of crizotinib and ruxolitinib disrupts this interaction. Furthermore, RON knockdown suppresses proliferation and activation of JAK/STAT signaling in JAK2-mutated cells, and RON deletion in a JAK2V617F mouse MPN model decreases the disease burden. We also observed RON hyperactivation in MPN patient cells, suggesting that RON may be an important target of crizotinib in MPN. In summary, we demonstrate that crizotinib has preclinical efficacy in MPN patient cells, JAK2-mutated cell lines, and a JAK2-mutated mouse model, and that the combination of crizotinib with JAK inhibitors suppresses JAK inhibitor persistence. Our work suggests that crizotinib should be investigated for the treatment of patients with MPN.

Efficacy of WWQ-131, a highly selective JAK2 inhibitor, in mouse models of myeloproliferative neoplasms

Hyperactivation of the Janus kinase 2 (JAK2) signaling pathway leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 can be used as an effective strategy for the treatment of MPNs. Here, our study indicated that WWQ-131 was a highly selective JAK2 inhibitor (IC50 =2.36 nM), with 182-fold and 171-fold more selective to JAK1 and JAK3, respectively. In JAK2V617F-dependent cell lines, WWQ-131 efficaciously inhibited cell proliferation, induced cell cycle arrest at the G2/M phase and apoptosis, and blocked the aberrant activation of JAK2 signaling pathway. In a mouse Ba/F3_JAK2V617F driven disease model, WWQ-131 effectively suppressed STAT5 phosphorylation in spleen and liver, and inhibited Ba/F3_JAK2V617F cells spreading and proliferation in vivo. In addition, WWQ-131 suppressed rhEPO-induced extramedullary erythropoiesis and polycythemia in mice, as well as hematocrits and spleen sizes, especially had no effect on white blood cell count. Furthermore, WWQ-131 (75 mg/kg) exhibited stronger therapeutic effects than fedratinib (120 mg/kg) in these two MPN models. Taken together, this study suggests that WWQ-131 will be a promising candidate for the treatment of MPNs.

3044 – IP-10 LOSS MODULATES ERYTHROID CELL PRODUCTION IN MOUSE MODELS OF MYELOPROLIFERATIVE NEOPLASMS

The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs. The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs.

Inhibiting Glutamine Metabolism with CB-839 Reduces Erythrocytosis in MPN Mice

Metabolic reprogramming is one of the hallmarks of cancer, as these rapidly dividing cells need to adapt their metabolism to cope with an increased energy demand. We have previously showed that in mouse models of myeloproliferative neoplasms (MPN), JAK2-mutant cells display metabolic alterations, including increased oxidative phosphorylation and glycolysis. The glutamine-glutamate-alphaKetoglutarate (aKG) axis, besides fueling the Krebs cycle and anabolic processes, contributes to the synthesis of the heme precursor 5-aminolevulinic acid (5-ALA), thus making glutaminolysis a potential target for MPN therapy.Here, we examined the effects of pharmacological inhibition of the conversion of glutamine to glutamate by glutaminase 1 by Telaglenastat (CB-839) in a mouse model of JAK2-V617F driven MPN. We performed competitive transplantations of bone marrow co-expressing JAK2-V617F with a GFP-reporter and wildtype mice in a 1:10 mixture into lethally irradiated mice. At 8 weeks post-transplantation, the mice developed MPN phenotype and were randomized into 4 treatment arms. They were then treated for 6 weeks with vehicle, CB-839, 3PO (6-phosphofructo-2-kinase inhibitor) and the combination of CB-839 + 3PO. We found that hemoglobin levels were significantly reduced in CB-839 treated mice and were fully normalised in mice that received a combination of CB-839 + 3PO (Fig. 1A). Other peripheral blood cell counts did not change (not shown). Hypoglycemia was noted in the vehicle group, as previously described, but an increase in blood glucose levels was seen already after one week of treatment with CB-839. 3PO alone had less impact on hypoglycemia and mice receiving CB-839 + 3PO had similar values to CB-839 alone (Fig. 1A). In line with these findings, splenomegaly was also reduced (Fig. 1A).To characterise the changes in metabolic activity, we determined the metabolic profiles of unfractionated bone marrow (BM) cells using seahorse assays. As expected, basal and maximal glycolysis and oxidative phosphorylation were higher in vehicle treated MPN cells than cells from wildtype controls. However, when BM cells from CB-839 or CB-839 + 3PO groups were examined, they displayed a strong downmodulation of both glycolysis and oxidative phosphorylation, indicating that chronic exposure to CB-839 reprograms the metabolic machinery by reducing not only the basal glycolytic and respiratory levels, but also the maximal capacity (Fig. 1B).Glutamine is the primary source of aKG needed to produce the heme precursor 5-ALA, and hemoglobin synthesis consumes the majority of ATP in erythroid progenitors. We therefore hypothesize that 5-ALA shortage caused by CB-839 limits hemoglobin production, leading to reduced catabolic activity and drop in ATP demand. To evaluate this model, we examined the stages of erythroid differentiation from vehicle or treated mice. Early and intermediate erythroid cells were enriched at the expense of later stages in CB-839 and CB-839+3PO treatment arms (Fig. 1C). The accumulation of erythroid precursors at the stage before hemoglobin is assembled supports the hypothesis that targeting glutamine usage impairs excessive erythropoiesis by limiting hemoglobinization rate.Our data show that inhibiting glutaminolysis ameliorated MPN phenotype and that the combination with the glycolytic inhibitor 3PO was able to induce complete hematological response. Since CB-839 is already in clinical trials for other cancers, our data suggest that it could also be tested for treatment of patients with polycythemia vera. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RON Kinase Is a Novel Therapeutic Target for Philadelphia-Negative Myeloproliferative Neoplasms

The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown.We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D).To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target.To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. [Display omitted] DisclosuresHalmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

Inhibition of SHP2 Ameliorates JAK2V617F-Induced Myeloproliferative Neoplasms

The oncogenic JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and advanced PV. Although ruxolitinib treatment can reduce splenomegaly and alleviate constitutional symptoms, it does not induce disease remission or cure bone marrow (BM) fibrosis in patients with MPN/MF. Thus, there is a critical need to identify new therapeutic target and develop novel targeted therapies for MPN/MF. We found that SHP2, a protein tyrosine phosphatase that positively regulates growth factor and cytokine signaling, is constitutively hyperphosphorylated in mouse and human MPN hematopoietic cells expressing JAK2V617F. We found that genetic ablation of SHP2 markedly inhibited the development of PV disease and BM fibrosis in Jak2V617F knock-in mouse models of MPN. So, we hypothesize that targeting of SHP2 alone or in combination with JAK2 inhibition might be useful for treatment of JAK2V617F-evoked MPN. In this study, we investigated the efficacy of SHP2 inhibition using an allosteric SHP2 inhibitor, SHP099, in hematopoietic cells and progenitors expressing JAK2V617F and a Jak2V617F knock-in mouse model of MPN.Treatment of SHP099 alone significantly reduced proliferation of murine BA/F3 cells expressing JAK2V617F. SHP099 treatment also significantly inhibited human JAK2V617F-positive UKE-1 cells. However, SHP099 only modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. SHP099 treatment also resulted in marked apoptosis in BA/F3-EpoR-JAK2V617F and UKE-1 cells but not in wild-type JAK2 expressing BA/F3-EpoR cells. We also found that SHP099 and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, SHP099 significantly inhibited hematopoietic progenitor colony growth in MPN/MF patient CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F (Jak2 VF/+) induces a PV-like disease, homozygous Jak2V617F (Jak2 VF/VF) expression promotes rapid progression to myelofibrosis. We utilized heterozygous Jak2V617F mice to test the in vivo efficacy of SHP099 against PV. Treatment of SHP099 significantly reduced the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in heterozygous Jak2V617F (Jak2 VF/+) mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors (HSPC) and myeloid precursors (Gr-1 +/Mac1 +) in the BM and spleens of Jak2V617F mice. Erythropoietin (Epo)-independent erythroid colony formation in the BM and spleens, a hall-mark feature of PV disease, was observed in Jak2V617F mice. Treatment of SHP099 markedly inhibited the Epo-independent CFU-E colonies in the BM and spleens of heterozygous Jak2V617F mice.We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using our homozygous Jak2V617F (Jak2 VF/VF) mice. SHP099 treatment alone or in combination with ruxolitinib significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in Jak2 VF/VF mice. Treatment of SHP099 or SHP099/ruxolitinib combination also significantly reduced the HSPC and myeloid precursor cells in the BM and spleens of Jak2 VF/VF mice. Histopathologic analyzes revealed extensive BM fibrosis in vehicle-treated Jak2 VF/VFmice. SHP099 treatment alone caused marked reduction of BM fibrosis in Jak2 VF/VF mice whereas combined treatment of SHP099 and ruxolitinib almost completely inhibited BM fibrosis in Jak2 VF/VF mice. Additionally, we performed competitive BM transplantation assays using Mx1Cre; Jak2 VF/VF GFP+ and WT mice BM cells followed by drug treatments. We observed that combined treatment of SHP099 and ruxolitinib significantly reduced the percentage of Jak2V617F mutant GFP+ LSK and myeloid cells in the BM of chimeric mice, suggesting that SHP099 and ruxolitinib combination preferentially inhibits oncogenic Jak2V617F mutant hematopoietic progenitors. Overall, our results suggest that SHP099 alone or in combination with ruxolitinib may have therapeutic potential against PV and MF and support the clinical investigation of SHP2 inhibitor in patients with PV/MF. DisclosuresNo relevant conflicts of interest to declare.

Telomerase Inhibition Impairs Self-Renewal of ß-Catenin Activated Myeloproliferative Neoplasm Progenitors

IntroductionChronic phase myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML) and myelofibrosis (MF), arise from hematopoietic stem cells. However, they harbor varying propensities to undergo blast crisis (acute leukemic) transformation based on their capacity to give rise to tyrosine kinase inhibitor (TKI) resistant myeloid progenitors that activate self-renewal pathways, such as Wnt/b-catenin signaling. Because b-catenin has been reported to transcriptionally regulate human telomerase reverse transcriptase (hTERT), we investigated the capacity of a telomerase complex inhibitor, imetelstat, to prevent malignant progenitor self-renewal. The telomerase complex consists of hTERT, an RNA template subunit (TERC), and a protective shelterin scaffold. Imetelstat is a novel, first in class covalently lipidated 13-mer oligonucleotide telomerase inhibitor with clinical activity in myeloid malignancies. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms such as MF. However, the role of imetelstat in selectively eradicating self-renewing MPN progenitors has not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized MPN progenitor primagraft studies to investigate the ability of imetelstat to selectively inhibit malignant progenitor self-renewal at doses that spare normal progenitors as well as to determine the mechanism of action.Methods and ResultsWhen FACS-purified progenitors from human blast crisis (BC; n=9) were compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples, RNA-seq revealed upregulation of key genes in the TERT interactome suggesting a role for TERT activation in MPN progenitor blast crisis transformation. Human MPN progenitor supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 µM significantly inhibited (p <0.001, ANOVA) in vitro replating of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Also, imetelstat (5 µM) significantly reduced replating of MF (n=9) compared with normal progenitor (n=4) samples. Treatment of humanized mouse models, established with four different BC CML samples, with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in both bone marrow and spleen malignant progenitor burden compared with mismatch controls (p=0.003, and p=0.009, t test), and a significant reduction in human CD45+ cells in the splenic niche compared with mismatch controls (p=0.03, t test). In a MF model established in NSG-S mice, imetelstat also reduced human CD45+ cell engraftment (p=0.03, t test). Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat treated MF cells (p=0.18) and CML cells (p=0.4) compared with mismatch control. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human progenitors after imetelstat treatment (p=0.02) compared with vehicle control.ConclusionsNiche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated MPN progenitors to imetelstat in both in vitro and in vivo humanized pre-clinical MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat.Disclosures: This work was funded by the Moores Family Foundation, the Koman Family Foundation, the California Institute for Regenerative Medicine (DR1-01430), Janssen Research & Development, LLC, and by NIH NCI R21CA189705. DisclosuresHuang:Janssen Research & Development, LLC: Employment.

Niche-Targeted Clinical Grade Immunohistochemistry Reveals Heterogeneous Bone Marrow Neuropathy and Novel Stromal Patterns in Primary Myelofibrosis Patients

Regulation of the hematopoietic stem cell (HSC) niche includes elements of the nervous system, immune system as well as the vasculature and associated stroma. Evidence in murine models demonstrate microenvironmental influence on HSC and hematopoiesis however correlative clinical and translational data remain scarce. The sympathetic nervous system may contribute to the pathogenesis of myeloproliferative neoplasms (MPNs) mechanistically through effects on nestin+ mesenchymal stromal/stem cells. Temporally, bone marrow neuropathy may even precede overt MPN by promoting aberrant premalignant HSC expansion, at least in genetically engineered MPN mouse models. We hypothesize that clinical characterization of the bone marrow niche via conventional immunohistochemistry (IHC) on routine diagnostic trephine biopsies is feasible and may yield novel stromal architectural data supplementing classification and/or prognostic stratification of BCR-ABL1 negative MPN. Herein, we describe our initial results on normal bone marrows and patients with primary myelofibrosis (PMF) with a focus on the pan-neuronal marker (PGP9.5) and the stromal marker smooth muscle actin (SMA). Results: Bone marrow neuropathy, as assessed by loss of punctate or linear perivascular PGP9.5 reactivity around arterioles is highly variable at initial PMF diagnosis. This suggests that in PMF, bone marrow neuropathy may not be universal and if present may only occur in a subset of patients or affect only focal regions of the marrow. SMA staining reproducibly yielded 3 patterns of reactivity in PMF: paratrabecular, perivascular and interstitial. While perivascular SMA staining was observed in all cases of PMF, the interstitial SMA pattern exhibited both inter-patient and intra-patient variability. Intriguingly, SMA was capable of highlighting “hot” and “cold” spots in PMF marrows otherwise appearing homogeneous by conventional reticulin staining. These SMA patterns are presently being investigated in the context of clinical information and molecular profiling to further assess their prognostic relevance. From longitudinal biopsies, we show resolution of the PMF interstitial SMA pattern as early as 2 months post allo-hematopoietic stem cell transplant, far earlier than any reduction in reticulin fibrosis which suggests that SMA signal may have important biological implications - including as a biomarker of early response to JAK inhibition, superior to standard reticulin/collagen assessment. In summary, visualization of the bone marrow niche by routine clinical grade IHC is feasible and reveals stromal heterogeneity within WHO-defined PMF of potential interest for classification refinement, prognostication and therapeutic monitoring. DisclosuresTsui:Novartis: Honoraria. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding.

JAK2-V617F and interferon-α induce megakaryocyte-biased stem cells characterized by decreased long-term functionality

AbstractWe studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.

Mouse models of myeloproliferative neoplasms for pre-clinical testing of novel therapeutic agents.

Myeloproliferative neoplasms (MPN), are clonal hematopoietic stem cell (HSC) disorders driven by gain-of-function mutations in JAK2 (JAK2-V617F), CALR or MPL genes. MPN treatment options currently mainly consist of cytoreductive therapy with hydroxyurea and JAK2 inhibitors such as ruxolitinib and fedratinib. Pegylated interferon-alpha can induce complete molecular remission (CMR) in some MPN patients when applied at early stages of disease. The ultimate goal of modern MPN treatment is to develop novel therapies that specifically target mutant HSCs in MPN and consistently induce CMR. Basic research has identified a growing number of candidate drugs with promising effects in vitro. A first step on the way to developing these compounds into drugs approved for treatment of MPN patients often consists of examining the effects in vivo using pre-clinical mouse models of MPN. Here we review the current state of MPN mouse models and the experimental setup for their optimal use in drug testing. In addition to novel compounds, combinatorial therapeutic approaches are often considered for the treatment of MPN. Optimized and validated mouse models can provide an efficient way to rapidly assess and select the most promising combinations and thereby contribute to accelerating the development of novel therapies of MPN.

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

Abstract 3792: Imetelstat inhibits RNA-editing mediated myeloproliferative neoplasm stem cell self-renewal

Abstract Introduction: Myeloproliferative neoplasms (MPN) including blast crisis (BC) CML and myelofibrosis (MF) are characterized by the clonal proliferation of hematopoietic cell lineages, mostly in the marrow. BC CML gives rise to TKI resistant myeloid progenitors that activate Wnt/β-catenin signaling pathway. However, BCR-ABL TKI resistant BC CML exhibits a robust telomerase activity and presents at very low or undetectable level in normal cells, and telomerase plays a pivotal role in cancer cell growth and may serve as an ideal target for anticancer therapeutics. In addition, β-catenin had been reported to transcriptionally regulate human telomerase reverse transcriptase (TERT). Telomerase is composed of the enzymatic reverse transcriptase protein TERT and the RNA template TERC. Thus, we investigated the capacity of imetelstat, a telomerase inhibitor, which binds to the TERC subunit with high affinity to prevent self-renewal of malignant progenitors. Recent clinical trials showed early signs of efficacy in MF. But, it's role in selectively inhibiting leukemia stem cell (LSC) self-renewing in CML has not been elucidated. Methods and Results: Progenitors from BC CML were compared with chronic phase (CP) CML and primary normal samples, RNA-seq results revealed upregulation of TERT suggesting a role for TERT activation in BC CML progenitor transformation. Human MPN progenitor co-culture experiments revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 or 5 uM significantly inhibited (p &amp;lt; 0.001, ANOVA) in vitro replating of BC CML compared with aged bone marrow progenitors. Also, imetelstat (5 mM) significantly reduced replating of MF compared with normal progenitor samples. Treatment of humanized MPN mouse models, established with 5 different BC CML and 4 different MF samples, at 30 mg/kg of imetelstat, three times a week for 4 weeks resulted in a significant reduction of malignant progenitors and human CD45+ cells (p &amp;lt; 0.001, t test) in BC CML, and a significant reduction of human CD45+ cells (p&amp;lt;0.01, t test) in MF in both marrow and spleen, when compared with vehicle controls. FACS analysis revealed a significant reduction of activated β-catenin protein in BC CML engrafted human progenitors after imetelstat treatment (p &amp;lt; 0.01, t test) compared with vehicle control. In addition, a significant inhibition of TERT (p = 0.03) and TERC (p = 0.02) was observed in BC CML LSC isolated from imetelstat treated mouse marrow when compared with vehicle control. Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Recent RNA-sequencing analysis showed an increase in ADAR1 expression during MPN progression, and our RNA-seq analysis demonstrated a decreased ADAR1 gene expression (p &amp;lt; 0.005) as well as overall adenosine to inosine editing rates as measured by edits per million reads (p &amp;lt;0.005) following imetelstat treatment of BC CML engrafted mice when compared to mismatch control. Conclusions: Niche responsive interactions between the telomerase complex and the Wnt/β-catenin self-renewal pathway sensitize β-catenin activated MPN progenitors to imetelstat treatment in both the in vitro co-culture and in vivo humanized MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat. Citation Format: Wenxue Ma, Larisa Balaian, Cayla Mason, Raymond Diep, Jessica Pham, Qingfei Jiang, Jeremy Lee, Sheldon Morris, Phoebe Mondala, Ping Chen, Thomas Whisenant, Mary Donohoe, Benjamin Heyman, Edward Ball, Fei Huang, Catriona Jamieson. Imetelstat inhibits RNA-editing mediated myeloproliferative neoplasm stem cell self-renewal [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3792.

3016 – SECRETORY MPP3 REINFORCE MYELOID DIFFERENTIATION TRAJECTORY AND AMPLIFY MYELOID CELL PRODUCTION

Hematopoietic stem cells (HSC) and their downstream lineage-biased multipotent progenitors (MPP) tailor blood production and control myelopoiesis on demand. However, we are still lacking the resolution of myeloid differentiation trajectories and cellular heterogeneity in MPP populations. Here, we show that myeloid-biased MPP3 are composed of several molecular subsets, with a distinct population of myeloid-primed cells with high endoplasmic reticulum (ER) volume and FcgR expression. Interestingly, FcgR+/ERhi MPP3 secrete high levels of IL-6 and TNFa cytokines upon inflammatory stimuli, and have faster division rates than ERlo MPP3. Moreover, condition media containing cytokines secreted by stimulated MPP3 promotes myeloid cell production from naïve HSCs, MPP3, and MPP4. These results suggest that FcgR+/ERhi MPP3 represent a transitional population toward granulocyte/macrophage progenitor (GMP) commitment, which could function as a self-reinforcing amplification compartment for myeloid differentiation in stress conditions. We next use our inducible Scl-tTA:TRE-BCR/ABL (BA) mouse model of myeloproliferative neoplasm (MPN) to investigate their role in leukemia. We find a massive expansion of FcgR+/ERhi MPP3 associated with a molecular remodeling of the MPP3 compartment and constitutive secretion of a unique set of cytokines, distinct from the ones produced in stress conditions. In particular, Galectin-3 (Gal3) is highly expressed by leukemic MPP3, and BA mice deficient for Gal3 (BA:Gal3-/-) show significantly reduced numbers of FcgR+/ERhi MPP3 and GMPs, and delayed disease onset. These results demonstrate the importance of FcgR+/ERhi MPP3 in amplifying myeloid cell production. Together, they identify a novel regulatory function for a subset of secretory MPP3 that controls myeloid differentiation through lineage-priming and cytokine production.

JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms

AbstractIncreased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2–driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.