Myeloproliferative Neoplasms Cells Research Articles

Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

Despite the identification of driver mutations leading to the initiation of myeloproliferative neoplasms (MPNs), the molecular pathogenesis of MPNs remains incompletely understood. Here, we demonstrate that growth arrest and DNA damage inducible gamma (GADD45g) is expressed at significantly lower levels in patients with MPNs, and JAK2V617F mutation and histone deacetylation contribute to its reduced expression. Downregulation of GADD45g plays a tumor-promoting role in human MPN cells. Gadd45g insufficiency in the murine hematopoietic system alone leads to significantly enhanced growth and self-renewal capacity of myeloid-biased hematopoietic stem cells, and the development of phenotypes resembling MPNs. Mechanistically, the pathogenic role of GADD45g insufficiency is mediated through a cascade of activations of RAC2, PAK1 and PI3K-AKT signaling pathways. These data characterize GADD45g deficiency as a novel pathogenic factor in MPNs.

Abstract 1485: Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations

Abstract Changes in gene expression and subsequent editing patterns of APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) cytosine deaminases have been shown to be present in solid tumor cancer evolution, however, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation, especially in the hematopoietic niche. Building on advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C and an editase-mutated APOBEC3C in normal cord blood, healthy bone marrow, and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in hematopoietic cells. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. These overexpression studies show novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis, which can be cross-referenced to gene expression and mutation signatures seen in hematopoietic malignancies and solid tumor cancers. In addition to mutation patterns, the regulation of LINE-1 elements has been shown to be another important function of APOBEC3 enzymes. In these studies, we examine the effects of APOBEC3 overexpression on repetitive element expression in hematopoietic cell populations and have found that there are strong cell type and context patterns across the APOBEC3 family and between cell types within the hematopoietic system. We will use this comprehensive study of APOBEC3 deregulation to uncover predictive biomarkers of malignant transformation. Citation Format: Jane Marie Isquith, Thomas Whisenant, Jessica Pham, Ludmil Alexandrov, Catriona Jamieson. Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1485.

SHP2 inhibition displays efficacy as a monotherapy and in combination with JAK2 inhibition in preclinical models of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, are clonal hematopoietic neoplasms driven by mutationally activated signaling by the JAK2 tyrosine kinase. Although JAK2 inhibitors can improve MPN patients' quality of life, they do not induce complete remission as disease-driving cells persistently survive therapy. ERK activation has been highlighted as contributing to JAK2 inhibitor persistent cell survival. As ERK is a component of signaling by activated RAS proteins and by JAK2 activation, we sought to inhibit RAS activation to enhance responses to JAK2 inhibition in preclinical MPN models. We found the SHP2 inhibitor RMC-4550 significantly enhanced growth inhibition of MPN cell lines in combination with the JAK2 inhibitor ruxolitinib, effectively preventing ruxolitinib persistent growth, and the growth and viability of established ruxolitinib persistent cells remained sensitive to SHP2 inhibition. Both SHP2 and JAK2 inhibition diminished cellular RAS-GTP levels, and their concomitant inhibition enhanced ERK inactivation and increased apoptosis. Inhibition of SHP2 inhibited the neoplastic growth of MPN patient hematopoietic progenitor cells and exhibited synergy with ruxolitinib. RMC-4550 antagonized MPN phenotypes and increased survival of an MPN mouse model driven by MPL-W515L. The combination of RMC-4550 and ruxolitinib, which was safe and tolerated in healthy mice, further inhibited disease compared to ruxolitinib monotherapy, including extending survival. Given SHP2 inhibitors are undergoing clinical evaluation in patients with solid tumors, our preclinical findings suggest that SHP2 is a candidate therapeutic target with potential for rapid translation to clinical assessment to improve current targeted therapies for MPN patients.

The glutaminase inhibitor CB-839 targets metabolic dependencies of JAK2-mutant hematopoiesis in MPN

AbstractHyperproliferation of myeloid and erythroid cells in myeloproliferative neoplasms (MPN) driven by the JAK2-V617F mutation is associated with altered metabolism. Given the central role of glutamine in anabolic and catabolic pathways, we examined the effects of pharmacologically inhibiting glutaminolysis, that is, the conversion of glutamine (Gln) to glutamate (Glu), using CB-839, a small molecular inhibitor of the enzyme glutaminase (GLS). We show that CB-839 strongly reduced the mitochondrial respiration rate of bone marrow cells from JAK2-V617F mutant (VF) mice, demonstrating a marked dependence of these cells on Gln-derived ATP production. Consistently, in vivo treatment with CB-839 normalized blood glucose levels, reduced splenomegaly and decreased erythrocytosis in VF mice. These effects were more pronounced when CB-839 was combined with the JAK1/2 inhibitor ruxolitinib or the glycolysis inhibitor 3PO, indicating possible synergies when cotargeting different metabolic and oncogenic pathways. Furthermore, we show that the inhibition of glutaminolysis with CB-839 preferentially lowered the proportion of JAK2-mutant hematopoietic stem cells (HSCs). The total number of HSCs was decreased by CB-839, primarily by reducing HSCs in the G1 phase of the cell cycle. CB-839 in combination with ruxolitinib also strongly reduced myelofibrosis at later stages of MPN. In line with the effects shown in mice, proliferation of CD34+ hematopoietic stem and progenitor cells from polycythemia vera patients was inhibited by CB-839 at nanomolar concentrations. These data suggest that inhibiting GLS alone or in combination with inhibitors of glycolysis or JAK2 inhibitors represents an attractive new therapeutic approach to MPN.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms

Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was >100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was >9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

BCL-Xl Represents a Novel Therapeutic Target in Type 2 Mutant Calr-Driven Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal disorders that arise in the hematopoietic stem cell (HSC) compartment and that result in the excess production of mature blood cells of the myeloid lineage. All MPNs are unified by their reliance on the constitutive activation of the JAK/STAT pathway. Nevertheless, JAK inhibitors have proven insufficient to significantly improve disease outcome, highlighting the need to further understand the molecular mechanisms driving these diseases and to discover novel therapeutic targets. Calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, has been found to be mutated in ~40% of essential thrombocytosis (ET) and myelofibrosis (MF) cases. The most common mutations in CALR are type 1 (52 base pair deletion; CALRdel52) and type 2 (5 base pair insertion; CALRins5) mutations, which both confer distinct clinical and prognostic characteristics. We have previously demonstrated that mutant CALR-driven MPNs rely not only on aberrant JAK/STAT signaling but also on the upregulation of BCL-2 family proteins and activation of the unfolded protein response (UPR) pathway (ASH abstract #147859, 2021). Specifically, we have shown that CALRins5 mutations lead to activation of and dependency on the activating transcription factor 6 (ATF6) branch of the UPR. In parallel, we found that CALRins5 cells exhibit differential up-regulation of the anti-apoptotic protein BCL-xL, but not BCL-2 (ASH abstract #3588, 2021). Based on these findings, we hypothesized that the active form of ATF6, which functions as a transcription factor, may directly transcriptionally activate BCL-xL leading to its up-regulation specifically in CALRins5 but not CALRdel52 MPN cells, and that the ATF6/BCL-xL axis may represent an important pro-survival mechanism that can be targeted for therapeutic gain in CALRins5 MPN patients. To test this hypothesis, we first validated that BCL-xL is significantly up-regulated in CALRins5 versus CALRdel52 and CALR wild type (CALRwt) human cell lines and peripheral blood mononuclear cells (PBMCs) from MPN patients. Because BCL-xL can also be regulated downstream of JAK/STAT signaling, we next tested whether the increased expression of BCL-xL in CALRins5 cells is due to ATF6 activation, JAK/STAT activation, or both. In cells treated with JAK inhibitor ruxolitinib, we found that while BCL-xL expression levels decreased nearly 90% in ruxolitinib treated CALRdel52 cells, this decrease was much more modest in CALRins5 cells, suggesting JAK/STAT activation is only partially responsible for BCL-xL up-regulation in CALRins5 MPN cells. Conversely, in ATF6 knockout cells, we found that deletion of ATF6 did not affect BCL-xL levels in CALRwt or CALRdel52 cells, but did significantly abrogate BCL-xL up-regulation in CALRins5 cells. This suggests that in CALRins5 cells, while JAK/STAT signaling contributes in part to BCL-xL up-regulation, ATF6 is the key mediator of BCL-xL expression. CUT&RUN studies further revealed that ATF6 directly regulates BCL-xL transcription. Lastly, we sought to test whether inhibition of BCL-xL alone is sufficient to induce apoptosis in CALRins5 cells. BH3 profiling studies revealed that CALRins5 cells are more primed for apoptosis via BCL-xL peptide antagonism compared to CALRdel52 cells. We next evaluated the activity of A-1331852, a BCL-xL selective inhibitor, alone or in combination with ruxolitinib, in human cell lines and PMBCs from MPN patients. We observed that CALRins5 cells displayed decreased viability in response to A-1331852 alone and in combination treatments, suggesting an increased sensitivity to BCL-xL inhibition. In conclusion, we demonstrate that CALRins5-driven MPN cells display an enhanced sensitivity to BCL-xL inhibition, which may represent an effective therapeutic approach for CALRins5+ MPN patients.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF > 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

Preclinical Studies Demonstrating Efficacy of Tasquinimod in Models of Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase

The alarmins, S100A8 (A8) and S100A9 (A9), are low molecular weight proteins belonging to the S100 protein family. Also called calprotectins, A8 and A9 can form homodimers or heterodimers and bind Ca++. A8 and A9 are secreted into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (Receptor for Advanced Glycation End products) and CD33. Intracellularly, they act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates NLRP3 inflammasome. A8 and A9 also amplify the inflammatory response through TLR4, and induce inflammatory cytokines, including TNFα and IL-6, through activation of NFkB and MAP kinase pathways. A8 and A9 are highly expressed in AML, especially myelomonocytic and monocytic AML, and are associated with poor prognosis. Through scRNA-Seq analysis, A8/A9 heterocomplex was shown to be highly expressed in myeloid and stromal cells and marked progression of bone marrow fibrosis in PMF (Primary Myelofibrosis). In the murine JAK2-V617F-driven PMF model, Tasquinimod (TM, Active Biotech), an orally active, quinoline-3-carboxamide linomide analogue immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR and RAGE receptors. Notably, TM treatment reduced myeloproliferation, splenomegaly and MF in JAK2-V617F mice. Therefore, based on these findings, in present studies, we determined in vitro and in vivo efficacy of TM against cellular models of advanced MPN cell lines and patient-derived (PD) CD34+ blastic phase (BP, > 5% blasts in PB) MPN cells. Treatment with TM (10 to 30 µM for 96 hours) induced loss of viability in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. RNA-Seq analysis showed that exposure to TM (20 µM for 16 hours) led to negative enrichment of the gene-sets of MYC and E2F targets, of mTORC1 and IL6-JAK-STAT3 signaling, and of inflammatory response in PD MPN-AML cells. This was associated with log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but upregulation of mRNA of GFI1 and p21. CyTOF analysis revealed that TM treatment significantly reduced expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67, but increased protein levels of GFI1, p21 and cleaved PARP in phenotypically defined PD, CD34+ MPN-BP cells expressing high levels of CLEC12A, CD99, CD123 but low expression of CD11b. Additionally, our findings showed that co-treatment with TM (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax induced synergistic lethality in advanced MPN-BP cells exhibiting delta synergy scores of >1.0 (by the ZIP method). Notably, our findings also demonstrated that treatment with TM (10 or 30 mg/kg/day, by oral gavage) for 8-weeks, significantly improved survival without inducing any toxicity in immune-depleted NSG mice engrafted with PDX cells of MPN-AML with mutations in CALR and TERT. In a separate experiment on the same PDX model, treatment with TM (30 mg/kg/day) also induced significantly greater survival advantage than treatment with ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) by oral gavage. These findings clearly demonstrate preclinical efficacy of TM in advanced MPN-BP cellular models and create the rationale to further interrogate the efficacy of TM alone and in combinations with current, front-line therapies for advanced MPN with excess blasts.

Galectin-1 Fuels Monocyte-Driven Hyperinflammation and Represents a Novel Therapeutic Target in Myeloproliferative Neoplasms

JDP and STO co-corresponding authors Previous studies by our group and others have demonstrated that monocytes play a key role in driving hyperinflammation in myeloproliferative neoplasms (MPNs), and that aberrant inflammatory cytokine signaling contributes to disease progression and poor prognosis in MPNs. To interrogate relationships between cellular sources and targets of inflammatory cytokines, we performed single cell RNA-seq (scRNA-seq) in PBMCs and progenitor cells from MPN patients and healthy controls. Monocytes from MPN patients displayed significant enrichment of inflammation-relevant genes as expected. Cell-cell communication networks inferred from expression of ligands and receptors predicted monocytes as a pivotal mediator of cell interactions. Further analysis revealed galectin signaling as one of the most robust input/output pathways for monocytes and significant enrichment of LGALS1 (galectin-1) expression in MPN monocytes. Galectins are a class of proteins that bind specifically to β-galactoside carbohydrates, such as N-linked or O-linked glycosylated proteins. Abnormal expression of galectins in various cancers has been found to mediate broad biological functions, including cell proliferation, apoptosis, adhesion, and inflammation. However, the mechanisms by which galectins contribute to MPN pathogenesis remain incompletely understood. To corroborate our sRNA-seq findings, we performed flow cytometry analysis which demonstrated elevated expression of galectin-1 in MPN patient CD14+ monocytes. Increased plasma levels of galectin-1 were also identified in MPN patients compared to healthy individuals. Higher galectin-1 protein levels in both Kit+ progenitor cells and CD11b+ myeloid cells from Jak2 V617F knock-in mice were also observed when compared to wild type mice. Additionally, galectin-1 expression was induced by MPL W515L and JAK2 V617F and inhibited by ruxolitinib in Ba/F3 cells. Thus, our results show enrichment of galectin-1 in both MPN patients and mouse models, with evidence of direct contribution from specific MPN driver mutations. Our scRNA-seq dataset analysis showed significant correlations of galectin-1 expression with inflammatory and oxidative phosphorylation (OXPHOS) genes in MPN patient monocytes. To investigate the effects of galectin-1 in monocyte activation, we incubated CD14+ monocytes from MPN patients with recombinant galectin-1 (rGal-1) and observed markedly stimulated secretion of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-8 and TNF-α. Furthermore, OTX008, a galectin-1 inhibitor, inhibited the transcription and secretion of inflammatory cytokines in MPN patient CD14+ monocytes and monocytic cell lines. Notably, both genetic and pharmacologic inhibition of galectin-1 reduced cellular ATP level and oxygen consumption rate, suggesting a metabolic reprogramming in MPNs. Although galectin-1 mediates broad biological functions, the molecular mechanisms by which galectin-1 regulates signaling pathways and transcription in MPN cells had not been delineated. Our scRNA-seq heterogeneity analysis suggested that genes in the PI3K-AKT-mTOR pathway were differentially expressed between galectin-1 high- and low-expressing monocytes. We therefore incubated both monocytic cell lines and monocytes from MPN patients with rGal-1 and observed stimulation of the PI3K-AKT-mTOR pathway, represented by increased levels of phosphorylated mTOR, AKT and S6. Consistently, genetic and pharmacologic inhibition of galectin-1 inhibited activation of the PI3K-AKT-mTOR pathway. In summary, our results demonstrate upregulation of galectin-1 in MPN monocytes, potentially due to activation of JAK2-driven signaling. We further demonstrate that galectin-1 fuels inflammation potentially via metabolic reprogramming of monocytes and activation of PI3K-AKT-mTOR signaling. Our data also suggest galectin-1 as a putative therapeutic target in MPNs.

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.

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML

Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated epigenome/transcriptome/proteome that inhibits differentiation and induces leukemia transformation (sAML) and therapy-refractoriness in MPN with excess blasts (> 5% in PB) or MPN transformed to AML (sAML). Treatment with JAK inhibitor (JAKi), e.g., ruxolitinib, venetoclax or hypomethylating agents alone or in combination are ineffective in improving the poor survival in MPN-sAML. CDK7 is the catalytic component of the general transcription factor TFIIH recruited with RNA-pol II (RNAP2) to transcription start-sites to initiate mRNA transcription. By phosphorylating CDK9 and negative transcript elongation factors, CDK7 also enables productive transcript elongation of oncogenes involved in growth and survival. In MPN-sAML, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1, and MYC. By phosphorylating CDK1/2/4/6, CDK7 also induces cell cycle. RNA-Seq analysis in BEAT AML study showed significantly higher CDK7 expression in MPN-sAML compared to normal progenitor cells. DepMap data involving CRISPR-mediated gene-knockouts show CDK7 as a dependency in the MPN-sAML SET2 and HEL cell lines. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and clinical grade SY-5609, dose-dependently (20 to 250 nM) increased % G1 while reducing the % of cell-cycle S phase SET2 and HEL cells. CDK7i treatment induced in vitro loss of viability in these cells, as well as in patient-derived (PD), CD34+ MPN-sAML but not in normal CD34+ progenitor cells. CRISPR-mediated CDK7 depletion (over 85%) in HEL cells significantly reduced SY-5609-mediated loss of viability. Importantly, pre-treatment with SY-5609 significantly increased sensitivity to ruxolitinib-induced loss of viability in JAKi-resistant SET2 and HEL cells, suggesting that CDK7 inhibition could overcome resistance to JAKi in MPN-sAML cells. RNA-Seq analysis conducted after SY-5609 treatment (100 nM for 16 hours) showed significant negative-enrichment of the gene-sets of MYC and E2F targets, cell-cycle checkpoints and of protein translation initiation/elongation, with log2 fold-decline in the mRNA levels of MYC, MYB, PLK1/4, KIT, CDK6, AURKA, PIM1 and CCND1, but upregulation of CDKN1A and HEXIM1. Following SY-5609 treatment of HEL cells, mass spectrometry demonstrated significant log2 fold-decline in c-Myc, c-Myb, RUNX1, PLK1, PIM1, but increase in p21, CASP9, BAD, DAP and TGFβ1 protein levels. CyTOF analysis demonstrated that SY-5609 treatment markedly reduced c-Myc, RUNX1 and MCL1, while increasing TP53, p21, and cleaved PARP proteins levels in MPN-sAML stem-progenitor cells defined by high expression of CD34, CLEC12A, CD123, CD99 but low expression of CD11b. Co-treatment with SY-5609 and ruxolitinib induced synergistic loss of viability in HEL, SET2 and PD MPN-sAML cells (n = 5) (delta synergy scores > 1.0 by the ZIP method). A CRISPR screen in SET2 and HEL cells targeting epigenetic regulators revealed significant log2 fold-decline in gRNA targeting BRD4, CBP and p300, highlighting them as dependencies. Consistent with this, co-treatment with SY-5609 and the BETi OTX015 was synergistically lethal in SET2, HEL, mouse MPN (JAK2-V617F plus TP53 loss) cells and PD MPN-sAML cells (n=5). SY-5609 treatment also exerted synergistic lethality with the BETi pelabresib or BD2-selective BETi ABBV-744 or the CBP/p300 inhibitor GNE-049 in MPN-sAML cells. In the xenograft model of HEL-Luc/GFP cells in NSG mice, monotherapy with SY-5609 (1.5 or 3.0 mg/kg/day, by oral gavage), vs vehicle control, significantly reduced the MPN-sAML burden and improved survival, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with SY-5609 and OTX015 (30 mg/kg/day by oral gavage) reduced more MPN-sAML burden and significantly improved survival in a HEL-Luc/GFP xenograft model without inducing toxicity. These findings demonstrate promising preclinical activity of CDK7 inhibition against the cellular models of MPN-sAML, supporting the rationale to further evaluate in vivo efficacy of CDK7i-based combinations against advanced MPN with excess blasts or MPN-sAML.

Methods to Elucidate Hematopoietic Clonal Organization in Bone Marrow

Every tissue in the human body becomes a mosaic of somatic mutations throughout life. Each cell in the body contains a record of the genetic alterations occurring from conception until that individual cell came into existence through cell division. Most of these changes are benign, but cells with alterations that endow a selective advantage can preferentially divide and expand. Already, this process has been described in a diverse set of phenotypically normal tissues including skin, esophagus and colon (Colom et al., 2021; Lee-Six et al., 2019; Martincorena et al., 2015). In these solid organs, clonal mutations are geographically constrained within contiguous regions of tissue. In the blood, these somatic mutations are termed clonal hematopoiesis (CH) (Steensma et al., 2015). Previous work has extensively characterized the burden of CH in humans, showing an increased prevalence with age and increased risk for hematologic malignancy and all-cause mortality (Genovese et al., 2014; Jaiswal et al., 2014; Young et al., 2016; Young et al., 2019). Despite our detailed understanding of CH in the blood, little is known about how these hematopoietic clones exist in the bone marrow (BM) and how they interact with the BM niche. We hypothesize that the spatial restriction of clones observed in solid organs also exists in the BM. To characterize the spatial and clonal organization within the BM niche, we combined single cell proteogenomics with novel methods for spatially aware variant detection. We characterized the BM clonal organization of a patient with polycythemia vera (PV)-a myeloproliferative neoplasm (MPN). MPNs are blood disorders characterized by abnormal hematopoiesis and an increased risk of transformation to aggressive leukemia. Studying a patient with PV was ideal as this is an oligoclonal disease, and we had prior knowledge that the patient's disease was driven by a large JAK2 V617F mutated clone and a presumed DNMT3A R882Cmutated subclone. Single BM cells were prepared for proteogenomics using the Tapestri platform (Mission Bio). From the BM aspirate, 1,486 individual live BM cells were assayed using the TotalSeq-D Heme Oncology Cocktail V1.0 (BioLegend) for immunophenotyping and the 45 gene myeloid panel (Mission Bio) for genomic characterization. Analysis identified the known JAK2 V617F and DNMT3A R882C mutations, and previously unknown TET2 L1081* and GNAS R844H mutations. The single cell assay disentangled the clonal complexity of this patient's disease-a large JAK2 V617F homozygous clone, a small TET2 L1081* heterozygous subclone, a small JAK2 V617F heterozygous clone, and independent GNAS R844H heterozygous and DNMT3A R882C heterozygous clones. Proteomic analysis revealed that the cells clustered into four groups including; B-lymphocytes marked by CD19, T-lymphocytes marked by CD3, the presumptive MPN cells highly expressing CD141 (thrombomodulin) and CD71 (transferrin receptor 1), and a mixed population of largely CD38 positive hematopoietic progenitors. To further characterize the spatial organization of these hematopoietic clones in the BM, the patient's femur head was sliced into 2mm sections using a band-saw and then further partitioned with hand tools into approximately 400 BM fragments, of which 96 were selected for droplet digital PCR (ddPCR) targeting the mutations identified by single-cell and bulk sequencing. The ddPCR assay identified each of these mutations in all assayed BM fragments, albeit at a wide range of variant allele fractions (VAFs). Interestingly, this suggested a heterogeneous distribution of somatically mutated clones spatially in the BM. Clustering of these bone marrow fragments identified regions marked by high JAK2 V617F and TET2 L1081* VAF (cluster 2), high JAK2 V617F VAF alone (cluster 3), and lower VAF for any mutations (clusters 1 and 2; Figure 1). These findings provide a comprehensive characterization of spatial organization and clonal hierarchy within the BM. Surprisingly, the clonal mutations were not well mixed in the BM with regions of high clonal diversity millimeters away from regions with a paucity of these mutated clones. Future work will expand this platform to describe BM clonal architecture in a larger set of bone marrow specimens and a wide variety clinical scenarios to help further explain the role of the BM niche in CH, MPNs and leukemia.

Glutamine Metabolism Is Altered in Myeloproliferative Neoplasms and Represents a Potential Novel Therapeutic Target

Cancer cells exhibit metabolic reprogramming to ensure sufficient production of macromolecules used to promote cell growth and proliferation. Previous studies have shown that proliferating cancer cells rely significantly on glutamine metabolism as a source of nitrogen and carbon to facilitate nucleotide and lipid biosynthesis, respectively, and to generate metabolites and/or maintain redox homeostasis. Despite this phenomenon being well characterized in many different solid and blood cancer types, very few studies have investigated the metabolic landscape of myeloproliferative neoplasms (MPNs). Glutamine catabolism begins via the uptake of glutamine through sodium-dependent transporters such as SLC1A5, and its transport to the mitochondria where it then undergoes deamination by glutaminase (GLS1) to produce glutamate. This conversion by GLS1 is the rate limiting step in the central glutaminolysis axis, making it a preferential point of study and therapeutic targeting. Indeed, many studies have explored the glutaminolysis pathway in a variety of cancers to decipher potential targets for treatment, but these studies have been extremely limited in MPNs and the potential mechanisms with respect to MPN driver mutations (CALR, JAK2, MPL) and their differential utilization of glutamine have yet to be fully explored. One previous study demonstrated that in JAK2V617F mutant-Ba/F3 cells, GLS1 expression was significantly increased and that GLS1 inhibition served as a promising intervention to suppress colony formation in patient samples but not healthy donors. To more comprehensively decipher the role of glutamine metabolism in MPNs driven by all three driver mutations ( CALR, JAK2, and MPL), we performed RNA-sequencing analysis on peripheral blood mononuclear cells (PMBCs) from MPN patients and found that genes involved in glutamine metabolism are significantly altered across the board. In contrast to the previous study mentioned above, our data demonstrate that GLS1 is not significantly up-regulated, and that MPN PBMCs are not sensitive to GLS1 inhibition. Rather, we characterized another key enzyme in glutamine metabolism, glutamine synthetase (GS) as a molecular and metabolic dependency in MPNs. GS catalyzes the reversion of glutamate into glutamine and serves to regulate nitrogen metabolism, support nucleotide biosynthesis, and most importantly synthesize glutamine to satiate the demand caused by the cancer cells' affinity for high glutamine levels. We found that GS is significantly up-regulated at the mRNA, protein, and activity level in MPNs driven by all three driver mutations, and that genetic and pharmacological inactivation of GS abrogates MPN cell proliferation in vitro. In summary, we have demonstrated that GLS1 may not be an effective therapeutic target for MPNs, and instead characterize GS as a novel dependency and potential point of therapeutic intervention.

DUSP1 Specific Protac Selectively Targets the JAK2 V617F Expressing Cells

Constitutive JAK-STAT signaling resulting from mutations in JAK2 V617F and certain growth-factor receptors has provided a strong rationale for targeting JAK2 kinase activity in myeloproliferative neoplasms (MPNs). Although several JAK2 inhibitors have been approved for MPN treatment, their clinical efficacy is limited to alleviating the constitutional symptoms associated with inflammatory cytokine signaling. These inhibitors are not cytotoxic and only exert a cytostatic response, failing to eradicate the malignant clone, induce molecular remission, or prevent progression to acute myeloid leukemia (AML). The inability of JAK2 TKIs to fully suppress the inflammatory cytokine overproduction, a key feature of MPN development and transition to AML, contributes to clonal persistence and TKI refractoriness. Recently, we reported that the cytokine signaling, in the context of JAK2 V617F signaling induces the expression of DUSP1, which enhances P53 degradation that leads to TKI refractoriness. Enhanced DUSP1 expression blocks P53 phosphorylation at Serine 15 (S15 phosphorylation prevents its interaction with MDM2) by inhibiting the P38 kinase activity. Consequently, reduced phosphorylation under JAK2 V617F signaling enhances P53 turnover. Deletion of DUSP1 enhances P38 kinase activity, leading to greater P53 stabilization and expression, which selectively induce cell death in JAK2 V617F expressing MPN cells while sparing normal cells. However, chemical inhibition of DUSP1 using a small molecule inhibitor (BCI) was ineffective in achieving synthetic lethality to JAK2 V617F cells, mainly due to pERK1/2 rebound caused by the off-target inhibition of DUSP6. Reintroduction of DUSP6 and BCI treatment restored JAK2 V617F clonal selectivity. This study provided the first mechanistic basis of TKI refractoriness in JAK2 targeted MPNs and suggested that selectively targeting DUSP1 could offer a stable or curative response in JAK2 V617F -driven MPNs. To address this, we developed a DUSP1-protac, a novel and highly potent DUSP1 selective PROTAC. Treatment of BaF3 cells expressing JAK2 V617F with DUSP1-protac resulted in dose- and time-dependent degradation of DUSP1, accompanied by increased P38 activity and P53 stabilization. The maximal level of DUSP1 degradation (Dmax) achieved was 95% with a DC50 of 70 nM/L within 24 hours. Pre-treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, reversed DUSP1 degradation, confirming that degradation occurs via the 26S-proteasome. As expected, DUSP1-protac induced cytotoxicity specifically in JAK2 V617F expressing cells, with a cellular IC50 of 50 nM. No toxicity was observed in BaF3 cells or those expressing MPL or CALR variants at concentrations up to 1000 nM, confirming its selectivity. In conclusion, our data suggest that DUSP1-protac is an exceptionally potent first-in-class DUSP1 selective PROTAC. It exerts a cytotoxic response specifically in JAK2 V617F expressing cells by enhancing P53 expression. Cellular uptake experiments and efficacy studies in primary cells are currently underway and will be presented in subsequent reports. The development of DUSP1-protac represents a promising approach for targeting JAK2 V617F -driven MPNs, offering potential benefits in achieving a stable or curative therapeutic response.

Leukemic Driver Clones Can Arise Independently and Outcompete MPN Cells in Post-MPN Secondary AML

The JAK2V617F variant is an acquired somatic mutation recurrently found in myeloproliferative neoplasm (MPN) patients and is used as a diagnostic marker. While considered a strong MPN driver mutation, this variant is sometimes absent in secondary acute myeloid leukemia (sAML) samples which transform from antecedent JAK2-mutant MPNs. The transformative evolution of MPN to sAML is thought to be driven by acquisition of additional genetic mutations affecting epigenetic modifiers (e.g. TET2), tumor suppressor genes (e.g. TP53), and splicing regulation, but the contribution and sequence of onset for these specific genetic events is not well understood. It is currently unknown why or how patients with a JAK2-mutant MPN may develop a JAK2 wild-type sAML. This transformation sequence is coupled with very limited treatment options and a survival of less than 6-months. Elucidating the evolutionary trajectory of secondary leukemic mutation occurrence is paramount for disease monitoring and early detection. To understand this evolutionary trajectory, we employed single cell proteogenomic analysis for three separate paired MPN patient samples (2 MF; 1 PV) who progressed to sAML stage. Clinical sequencing data indicated presence of the JAK2 V617F mutation in all three samples at the MPN stage whereas this mutation was not detected at the sAML stage. We employed the MissionBio Tapestri platform to delineate the clonal trajectory of transformation to determine if sAML and MPN driver clones share a common founder clone or if they arise through independent, non-clonally related founders. Utilization of this single-cell platform enabled us to investigate two potential models of transformation. First, two distinct mutation phases of MPN to sAML occur wherein the JAK2 -mutant clone and sAML driver mutations originate form a shared founder clone. In this model, an MPN phenotype driven by JAK2-mutant clones arises in patients first, followed by the sAML disease stage driven by leukemic mutations. An alternative tested model is that MPN and sAML originate as separate clones wherein the JAK2 mutant hematopoietic stem cells (HSCs) give rise to an MPN phenotype first, while the independent sAML clone(s) grow slowly yet separately in the background of the existing MPN. Both models provide a plausible explanation which results in leukemic blast stage clones harboring leukemic mutations but lacking the MPN driving mutation JAK2V617F. Single-cell analysis detected the JAK2V617F mutation in both the MPN (MF) as well as the sAML stage in one of the paired patient samples in which JAK2 was previously reported as wild-type at the sAML stage. This sample also contained a RUNX1 mutation at the sAML stage as well as a clonally unrelated ATM mutation (Figure 1A). The single-cell data for the remaining two paired patient samples (1MF; 1PV) demonstrated that pre-leukemic driver mutations were not detected within the JAK2-mutant clones at the MPN stage or during a middle time-point stage before AML diagnosis. Single-cell sequencing from the sAML time point for both samples demonstrated loss of the JAK2-mutant clone (verified by ddPCR analysis), amidst presence of leukemic driver mutant clones (Figure 1B). This clonal hierarchy suggests that the leukemic mutations were not present within the JAK2-mutant clones during the MPN stage and arose in the background of the MPN where they outcompeted the MPN driver clone during sAML transformation. Further, this hierarchy suggests that the AML driver mutations do not share a clonal intermediate ancestor with the JAK2 mutant cells which drove the MPN. These data demonstrate that one clonal trajectory of JAK2-mutant MPN to JAK2 wild-type sAML is through independent leukemic clones arising in the background of MPN and outcompeting the JAK2-mutant clones. In these instances, it is more appropriate to view these patients as de novo AML cases arising in a background of pre-existing MPN, which could influence treatment decisions and disease surveillance.

Divergent Functions of ERK2 Substrate Binding Modalities in Myeloproliferative Neoplasm

Introduction: JAK2-V617F driven myeloproliferative neoplasms (MPNs) are intractable blood disorders marked by the elevated production of erythrocytes and myeloid cells in the bone marrow. While current JAK2 inhibitors offer some respite, they fail to eradicate the mutated cell population due to acquired treatment resistance. Treatment resistance arises from compensatory activation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway; however, even combination therapy targeting both JAK2 and the MAPK signaling has led to drug resistance. Instead of attempting to inhibit kinase activity, we propose to attenuate ERK2 substrate interactions that drive cancer. We have discovered that the two substrate binding domains of ERK2, D and DBP, play opposing roles in MPN pathogenesis. The DBP domain attenuates MPN pathogenesis and promotes oncogene-induced-senescence (OIS) through physical association with the DEF motif of Egr1, while the D-domain promotes progression, through Polq induction. Methods: To study the divergent effects of ERK2 DBP and D domain on MPN progression, we introduced single point mutations in the substrate binding domains of ERK2 using CRISPR-Cas9. The expression of mutant alleles of ERK2 is conditionally activated by excision of the floxed allele using Vav-Cre. Similarly, we constructed a CRISPR knock-in mouse model, Egr1-Y253A, to examine the role of the Egr1 DEF motif in regulating OIS. To assess the response of individual malignant clones to Polq inhibitor (Polqi), we employed single-cell NGS and the Tapestri platform to detect clinically relevant mutations in 54 genes associated with myeloid neoplasms. For the inhibitor studies, we used human MPN cell lines and independent MPN patient cohorts. Results: Ectopic expression of JAK2 V617F in DBP-inactivated progenitors enhanced their clonogenic potential and abrogated OIS in vitro. This occurs because mutating the ERK2-DBP domain prevents interaction with the senescence-inducing transcription factor, Egr1. Egr1-Y253A mutant progenitors display a similar phenotype in colony formation and OIS function supporting our previous findings that physical association with the DBP domain is required for senescence induction. Because disabling the DBP domain accelerated MPN progression (Zhang et al, Blood, 2022), it is suggested that the D domain promotes progression. Induction of Polq protein is observed with ectopic expression of oncogenic MPN drivers in a myeloid cell line, which is inhibited by blocking ERK activity. The increased expression of Polq is crucial for MPN growth and survival, as the growth of HSPCs transduced with MPN oncogenic drivers is attenuated in the absence of Polq. Notably, Polq expression is upregulated in HSPCs from ERK2-DBP mutant mice, both before and after transduction with oncogenic JAK2 and CALR, thereby suggesting that the remaining substrate interaction domain, ERK2-D, is responsible for its induction. Interestingly, the ERK2-D domain not only interacts with Polq but may also regulate its expression through ubiquitin-dependent degradation involving the E3-ubiquitin ligase, c-CBL. D-domain inhibitor (76) substantially reduced disease burden in a xenotransplantation model of SET2 MPN line. We found that targeting the ERK2-D domain substrate, Polq, was similarly effective. Lin-CD34+ MPN HSPC were more sensitive to Polq inhibitor, ART558, compared to normal implicating Polq as a therapeutic target in MPNs. Results from single cell sequencing analysis of a triple-negative MPN which transitioned to AML, indicated a high degree of clonal variation in sensitivity to Polqi, with WT1 clones being sensitive and NRAS mutant clones exhibiting resistance. Thus, a combination of Polqi + ERK2-Di might exhibit a synergistic anti-MPN effect by attenuating the resistance mechanism of malignant subclones. As an initial test of this possibility, we treated SET2 cells with Polqi + ERK2-Di combination therapy, which revealed a synergistic response. Conclusion: The findings suggest that a combination therapy, such as Polqi + ERK2-Di, may be a promising strategy to overcome treatment resistance arising from clonal variations in sensitivity to single agents. By targeting both the D domain and inhibiting the downstream substrate Polq, a synergistic anti-MPN effect may be achieved, potentially offering a more effective therapeutic approach for patients with MPNs.

A Dual-Specific Inhibitor of Rock/Aurk, RR-1752, for Primary Myelofibrosis

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by driver mutations in JAK2, CALR, and MPL resulting in constitutive activation of JAK-STAT signaling. Abnormalities in megakaryocyte (MK) numbers and morphology are a common feature of MF where abnormal MKs are associated with bone marrow fibrosis and pro-inflammatory cytokine release. Therapies that target constitutive JAK-STAT signaling and atypical megakaryocytes could provide clinical benefit for patients with MF. ROCK and Aurora kinase (AURK) are overexpressed in MPN and linked to dysregulated MKs of patients with MPN. We therefore tested the activity of RR-1752, a dual-specific inhibitor of ROCK and AURK, in preclinical models of MF. RR-1752 acted as an inhibitor of pSTAT3 in IL-2 stimulated human PBMC 1h post stimulation and significantly inhibited phosphorylated myosin light chain 2 (pMLC2) in two human MPN cell lines, HEL-92.1.7 and UKE-1, confirming an on-target action. RR-1752 inhibited colony formation by HEL-92.1.7 and UKE-1, (IC 50 = 25.2 nM and 54.6 nM respectively). RR-1752 also induced cell cycle arrest in HEL-92.1.7 and UKE-1 specifically at the G2/M checkpoint. RR-1752 was also tested in a hematopoietic colony formation assays of MF patient derived CD34 + cells. RR-1752 led to concentration specific inhibition of MF patient derived CD34 + cell colony formation between 100-190 nM demonstrating relevant activity against patient-derived MF samples (51% inhibition at 190 nM as compared to vehicle control). These data were in line with flow cytometrically determined cell viability data of MF CD34 + cells cultured in the presence of varying concentrations of RR-1752 in liquid culture. The in vivo activity of RR-1752, was examined using the human HEL- 92.1.7 cell line, homozygous for both JAK2 V617F and p53 M133K in a mouse xenograft model. The effects of RR-1752 alone and in combination with ruxolitinib were compared to single agent ruxolitinib. The administration of RR-1752, 25 mg/kg PO daily resulted in a significant (p<0.01) improvement in median survival (31 days) compared to ruxolitinib alone, 45 mg/kg PO daily (21 days). RR-1752 5.0 mg/kg PO QD plus ruxolitinib 45 mg/kg PO QD also resulted in a significant improvement in survival (p<0.05) compared to single agent ruxolitinib but was inferior to single agent RR-1752 at 25mg/kg. The demonstrated activity in MF patient derived CD34 + cells combined with the significant improvement in survival compared to the approved drug ruxolitinib in an aggressive, JAK2/p53 mutant MF xenograft model provides a compelling case for its evaluation as a therapeutic modality in MF patients. Dose optimization of RR-1752 alone and in combination with ruxolitinib in the HEL-92.1.7 IV dissemination model as well as testing in the hMPL-W515L-evoked myelofibrosis model is ongoing. Conventional treatments for MF have largely focused on symptom management but RR-1752 treatment could result not only in beneficial symptom improvement but also important disease modifying potential by inhibiting JAK-STAT signaling, cell cycle progression and targeting dysregulated MKs.

Development of Resistance to Type II JAK2 Inhibitors in MPN Depends on AXL Kinase and Is Targetable.

Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Because clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC/RNA sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2,500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NOD/SCID gamma mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Because inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL/MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL/MAPK. We report on a novel mechanism of AXL/MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN.