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Chronic graft-vs-host disease (cGVHD) is a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HCT) in patients. This debilitating condition is characterized by chronic inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is one of the more severe forms of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy. Methods: To address the current gap in knowledge pertaining to the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on fresh patient biopsy specimens. In vivo studies were carried out by sub lethal irradiation of BALB.k recipients which underwent HCT from miAg-mismatched AKR/J donors. Recipient sclerodermatous-tissues were analyzed using FACS, IHC and IF staining. Human studies were conducted on (i) Primary samples from patients with severe sclGVHD using tissue microarrays (TMA) by Immuno-histofluorescence (IHF) and IF. (ii) Dermal fibroblasts from sclGVHD samples were subjected to ATACseq and ChiPseq CRISPR-Cas9 JUN deletion. (iii) Also, dermal fibroblasts from human scl-GVHD were implanted under the kidney capsule of NSG mice to study the effects of inhibiting pro-fibrotic pathways in vivo. Results: We show for the first time that in a mouse model of sclGVHD (male), recipients of female T-cell replete grafts developed severe scleroderma with massive skin thickening and collagen deposition. Fibroblasts strongly expressed JUN, which is part of AP-1, a transcription factor involved in the acute phase response that regulates gene expression in response to stimuli from cytokines, growth factors and pathogens. We have previously demonstrated JUN as a key player in the molecular pathogenesis of other fibrotic diseases (Wernig G et al. PNAS 2017, Cui et al. Nature comm. 2020, Lerbs et al. JCI i 2020). Likewise, CD47, an immune checkpoint protein that prevents removal of Mϕ, was strongly co-expressed in fibroblasts in sclGVHD - but not in the control mice (Fig A+B). Here we show that (i) In humans, (n = 45 sclGVHD patients), there is a strong expression and activation of JUN and CD47 in dermal fibroblasts which was not observed in control samples. Mixed inflammatory infiltrates were dominated by Mϕ and granulocytes. (ii) Isolated primary fibroblasts from fresh human sclGVHD skin biopsies analyzed for chromatin accessibility across the genome by ATAC-seq showed wide open accessibility to the JUN promoter, IL-6 promoter and CD47 enhancer and promoter indicating that they play a critical role in regulating the pathogenesis of sclGVHD. In contrast, normal fibroblasts displayed only minimal accessibility to the JUN promoter. We further validated our data using CRISPR-Cas9 knock-down studies on sclGVHD fibroblasts and show that the IL-6 promoter, enhancer and promoter of CD47 are regulated by JUN, with JUN deletion resulting in significant decrease in the promoter binding accessibility to IL-6 and CD47 (Fig C). Further, JUN activity appears to regulate key members of the Hh signaling pathway (GLI1, PTCH1 and PTCH2), as their chromatin accessibility was decreased with JUN deletion. These correlative findings were confirmed by JUN ChIP seq, an assay that identifies binding sites of DNA-associated proteins. (iii) To test our findings in vivo we established xenograft models of primary human sclGVHD by implanting cells under the kidney capsule of NSG mice.All treatments (except placebo) resulted in decreased fibrosis (Fig D), presumably by blocking the activation of JUN (pJUN) and its profibrotic downstream pathway members IL-6 and pSTAT3, as assessed by phospho flow. Conclusion: In our studies we demonstrate that in established SclGVHD, combinatorial therapy consisting of anti-CD47 antibody together with IL6 blockade has the highest potential to translate into a therapeutic intervention given its ability to be more effective than currently used antifibrotic and anti-inflammatory agents in clinic. The findings from our study are significant because we show an important mechanism underlying SclGVHD onset, identify a novel genetic signature that can be targeted, describe a new mouse model and a clinical assay that has a high throughput readout and suggest a treatment regimen for patients. Figure 1 Figure 1. Disclosures Arai: Magenta Therapeutics: Research Funding. Shizuru: Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020; Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board.

Understanding the contribution of abnormal genetic and epigenetic programs to acute myeloid leukemia (AML) is necessary for the integrated design of genetic and epigenetic targeted therapies. While epigenetic therapies have been used to treat AML, so far efficacy has been limited. To this point, the reversibility of epigenetic modifications in AML is poorly understood, as are the relative contributions of the leukemic genetic and epigenetic programs to disease pathogenesis. To investigate these questions, we sought to reprogram primary AML leukemic blasts into induced pluripotent stem cells (iPSCs) and assess the effect of epigenetic reprogramming on leukemic behavior. We generated iPSCs from several AML patients with 11q23/MLL translocations using classic iPSC reprogramming factors (Sox-2, Klf4, Oct4, and Myc). Reprogrammed AML cells generated iPSCs as indicated by classic pluripotent features including embryonic stem cell morphology, expression of pluripotent markers, and formation of teratomas in vivo. In addition, AML iPSCs retained all the karyotypic and genetic abnormalities of the original AML patient. Despite presence of these mutations, AML iPSCs were able to differentiate into multiple cell types with normal function. Thus, human AML cells can be epigenetically reprogrammed into a pluripotent state despite the presence of leukemic driver mutations. Surprisingly, when AML iPSCs were differentiated in CD43+45+ hematopoietic cells, a leukemia phenotype re-emerged. Differentiated hematopoietic cells from AML iPSCs exhibited exclusively granulocytic-monocytic differentiation in colony forming assays, and serial replating potential in contrast to control iPSCs. When transplanted into immunodeficient mice, hematopoietic cells from AML iPSCs formed aggressive myeloid leukemias as evidenced by peripheral blood and bone marrow CD33+ myeloid engraftment, enlarged spleens, secondary transplant potential, and death from fulminant leukemia. These data indicate that epigenetic reprogramming alone was insufficient to eliminate leukemic behavior. To further investigate these epigenetic changes during reprogramming, we performed DNA methylation and gene expression analysis using 450K BeadChip arrays and RNAseq, respectively. Unsupervised hierarchical clustering demonstrated clustering of undifferentiated AML iPSCs with control iPSCs. In contrast, hematopoietic cells differentiated from AML iPSCs clustered exclusively with primary AML cells. Through gene ontology enrichment analysis, hematopoietic differentiated AML iPSCs were enriched for hypomethylation and gene activation of hematopoietic and leukemogenesis gene sets, including MLL gene targets, as compared to undifferentiated AML iPSCs. These data demonstrate that DNA methylation and gene expression profiles are reset upon epigenetic reprogramming of AML cells, but re-emerge upon hematopoietic differentiation, coinciding with re-emergence of the leukemic phenotype. Lastly, we demonstrated that our AML iPSC model can be used to physically separate and functionally profile genetic subclones within an AML patient. In one patient, we identified distinct AML iPSC genetic subclones (KRAS wildtype and G13D mutant) that could be prospectively separated and demonstrated differential growth properties and therapeutic susceptibilities. To determine the clinical relevance of these subclones, we sequenced this patient at relapse. Strikingly, the KRAS mutant clone, which was dominant at diagnosis, was absent at disease relapse implying relative chemotherapy resistance and competitive outgrowth of the KRAS wildtype subclone. Indeed, in our AML iPSC model, the KRAS wildtype clone demonstrated increased resistance to cytarabine in colony assays. We then investigated in a cohort of AML patients whether cytarabine sensitivity differed between KRAS wildtype and mutant patients. Strikingly, KRAS AML patients were more resistant to cytarabine in vitro compared to KRAS G13D patients. Thus, our AML iPSC model predicted clonal relapse in this patient and identified differential subclonal sensitivity to chemotherapy as a mechanism for relapse. In summary, pluripotent reprogramming of AML resets leukemic methylation/gene expression patterns, facilitates clonal targeting, and predicts subclonal relapse. Disclosures Chao: Forty Seven Inc.: Employment, Equity Ownership, Patents & Royalties. Majeti:Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However, there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL, based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation, and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy, we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model, JAK2T875N induced a myeloproliferative disease characterized by features of AMKL, including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL.

An acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

Approximately 50% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) lack activating mutations in JAK2. Among these patients, ~10% harbor an activating mutation in the thrombopoietin receptor, MPLW515L. We have reported that expression of MPLW515L in a murine bone marrow transplant model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis, that are not observed in the JAK2V617F model. These observations provide an opportunity to assess the efficacy of small molecule JAK2 inhibitors on a myeloproliferative disease (MPD) induced by MPLW515L in vivo, and to determine whether such inhibitors attenuate thrombocytosis. We have tested EXEL-8232 for efficacy in suppression of thrombocytosis in vivo, and for its ability to attenuate JAK2V617F-negative MPD mediated by MPLW515L. EXEL-8232 is a potent small molecule inhibitor of JAK2 and is structurally similar to XL019, a compound currently in clinical trials for MF and polycythemia vera. EXEL-8232 is selective for JAK2 with a biochemical IC50 of 2 nM, and abolished constitutive phosphorylation of JAK2 and STAT5, as well as cytokine-independent growth, of Ba/F3 cells in vitro. After disease was established 12 days post-bone marrow transplantation, EXEL-8232 was administered for 28 days q12h by oral gavage at doses of 30mg/kg or 100mg/kg respectively. Animals treated with 100mg/kg normalized high platelet counts in excess of2 million/ml and normalized leukocytosis from a median of 134,000/ml in vehicle treated controls. Furthermore, drug treatment eliminated extramedullary hematopoiesis in the spleen, as well as bone marrow fibrosis. Of note, EXEL-8232 had no impact on erythrocytosis in diseased animals or in wild type controls, and wild type animals treated with either dosage of 30mg/kg or 100mg/kg did not develop thrombocytopenia. Consistent with these clinical responses, the surrogate endpoints for response to treatment included a reduction of genomic disease burden in the 100mg/kg treated arm (p

The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Mutations that result in constitutive tyrosine kinase activation occur in a wide spectrum of human malignancies, including acute myeloid leukemia (AML). Although activating mutations in the tyrosine kinases FLT3 and c-KIT occur in a significant proportion of patients with AML, the genomic events responsible for oncogenic signaling in most patients with AML have not been identified. To determine whether other aberrantly activated tyrosine kinases contribute to the pathogenesis of AML, we employed high throughput (HT) DNA sequence analysis to screen the exons encoding domains implicated in kinase activation (activation loop), and autoinhibition (juxtamembrane domains) in 85 tyrosine kinase genes in 192 AML patients without FLT3 or c-KIT mutations. The screen identified 34 non-synonymous sequence variations in 25 different kinases that had not been reported in single-nucleotide polymorphism (SNP) databases. These included a novel activating allele in FLT3, and a previously described activating mutation in MET (METT1010I). The majority of the novel sequence variants were cloned into their respective tyrosine kinase cDNA, and stably expressed in the factor-dependent Ba/F3 hematopoietic cell line. Apart from one FLT3 allele, none of the other novel variants showed evidence of constitutive phosphorylation by immunoblot analysis, and none transformed Ba/F3 cell lines to factor independent growth. These findings indicate that the majority of these alleles are not potent activators of tyrosine kinase activity in this cellular context, and that a significant proportion of non-synonymous sequence variants identified in HT DNA sequencing screens may not have functional significance. Although several explanations are possible for this observation, these data are consistent with recent reports that a significant fraction of such sequence variants are “passenger” rather than “driver” alleles, in cancer, and underscore the importance of functional assessment of candidate disease alleles.

Although acquisition of JAK2V617F mutation is an important pathogenetic event in many patients with polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), the genetic events that contribute to most cases of JAK2V617F-negative MPD are not known. We used high throughput DNA sequence analysis of cytokine receptors critical for erythroid, megakaryocytic, and granulocytic proliferation and identified a somatic mutation in the thrombopoietin receptor (MPLW515L) in patients with JAK2V617F-negative MF. Expression of MPLW515L transforms hematopoietic cells to cytokine-independent growth, and activated components of the JAK-STAT signal transduction pathway including JAK2, STAT5, STAT3, ERK and AKT. We compared the disease phenotype induced by MPLW515L with that we have previously reported for the JAK2V617F allele in a murine bone marrow transplant model of myeloproliferative disease. In Balb/C mice, expression of MPLW515L caused a rapidly fatal, fully penetrant MPD (median latency of ~18 days) that was characterized by marked leukocytosis and thrombocytosis, with average platelet count of 3.4 million/ul as well as bone marrow reticulin fibrosis and thrombotic complications. A similar phenotype was observed in MPLW515L C57/Bl6 recipients, but with a longer median latency of ~50 days. In a subset of C57/Bl6 mice, the acute MPD was followed by a progressive reduction in platelet count and an increase in bone marrow fibrosis that occurred over 3 months, and was reminiscent of human MF. These findings stand in contrast to MPD induced by the JAK2V617F allele in C57/Bl6 mice, in which there is marked erythrocytosis, but not thrombocytosis or thrombotic complications or leukocytosis. Analysis of myeloid progenitor populations using multiparameter flow cytometry demonstrated an expansion of the common myeloid progenitor (CMP), the granulocyte-monocyte progenitor (GMP) population, and the megakaryocytic-erythroid progenitor (MEP) population in MPLW515L-expressing bone marrow and spleen, with up to a 10-fold increase in CMPs and an 8-fold increase in MEPs, whereas there were no differences observed in the relative proportion of CMP, GMP or MEP populations in JAK2V617F mice. These data demonstrate important phenotypic differences between disease induced by the MPLW515L or JAK2V617F alleles in C57/Bl6 mice. These include (i) marked differences in the degree of leukocytosis, with MPLW515L induced marked leukocytosis compared with JAK2V617F and (ii) the effect on the megakaryocyte lineage in which JAK2V617F induces megakaryopoiesis, but with reduced megakaryocyte ploidy with no thrombocytosis or thrombotic complications, whereas MPLW515L enhances megakarypoiesis and induces thrombocytosis with thrombotic complications. These findings suggest that JAK2V617F and MPLW515L - each of which is characterized by activation of JAK-STAT signaling in hematopoietic progenitors - have qualitative and quantitative differences in their ability to impact proliferation and/or survival of hematopoietic progenitors that presumably are the consequence of differences in signal transduction, and result in disparate phenotypes.