Proposed model of efficacy of dual inhibitor of Tie2 and p38 MAPK in MDS and AML

Supp Fig 1 : SFRP1 methylation with coculture Supp Fig 2 : Validation of methylation changes with stroma and leukemic cell coculture Supp Fig 3: FRZB upregulation in MDS stroma after 5-Azacytidine treatment SuppFig 4: FRZB knockdown leads to partial inhibition of erythroid differentiation induced by treatment of MDS stromawith 5-Aza SuppFig 5: The expression of WNT signature genes in MDS and control marrow derived CD34+ cells

The MLL3 gene has been shown to be recurrently mutated in many malignancies including in families with acute myeloid leukemia. We demonstrate that many MLL3 variant calls made by exome sequencing are false positives due to misalignment to homologous regions, including a region on chr21, and can only be validated by long-range PCR. Numerous other recurrently mutated genes reported in COSMIC and TCGA databases have pseudogenes and cannot also be validated by conventional short read-based sequencing approaches. Genome-wide identification of pseudogene regions demonstrates that frequency of these homologous regions is increased with sequencing read lengths below 200 bps. To enable identification of poor quality sequencing variants in prospective studies, we generated novel genome-wide maps of regions with poor mappability that can be used in variant calling algorithms. Taken together, our findings reveal that pseudogene regions are a source of false-positive mutations in cancers

The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64–6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population. First responders to the World Trade Center disaster, who were exposed to particulate matter containing potential carcinogens, have a high burden of somatic mutations in blood cells, raising their risk for cancer and other diseases and highlighting the need for enhanced health screening of these individuals.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Key Points North American ATLL has a distinct genomic landscape with a high frequency of prognostic epigenetic mutations, including EP300 mutations. ATLL samples with mutated EP300 have compromised p53 function and are selectively sensitive to decitabine treatment.

Abstract Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastasis. To study this at a genome-wide level in melanoma, we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. Its expression was validated by IHC and RNA-FISH on primary tumors and by qPCR, Western blotting and FACS in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin's lymphoma. After analyzing our patient samples and cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELISA and qPCR pointing to a potential autocrine regulatory loop. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in 2D and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness suggesting a functional role for CSF-1R in melanoma. Treatment of melanoma with BRAF-V600E inhibitors is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and the rebound activation of the MAPK-pathway. We are suggesting a role for CSF1R in this process. In Western experiments, the rebound of phospho-ERK after BRAF inhibitor treatment was accelerated with the addition of CSF1R ligands, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing shRNA against CSF1R recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the receptor, as well as its maturation, and presentation on the cell surface. shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression in resistant tumors. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo.The CSF1R inhibitor, PLX3397 is currently in clinical trials for glioblastoma, prostate, breast cancers and other cancers. These data present a preclinical rationale for its study in malignant melanoma. Citation Format: Orsolya Giricz, Yongkai Mo, Kimberly B. Dahlman, Xiomaris M. Cotto-Rios, Chiara Vardabasso, Hoa Nguyen, Bernice Matusow, Matthias Bartenstein, Veronika Polishchuck-Lee, Douglas B. Johnson, Tushar B. Bhagat, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John M. Greally, Yiting Yu, Gideon Bollag, Paraic A. Kenny, Kith Pradhan, E. Richard Stanley, Emily Bernstein, Evripidis Gavathiotis, Brian L. West, Jeffrey A. Sosman, Amit Verma. Aberrant expression of CSF1R in melanoma is driven through an endogenous viral promoter and it contributes to malignant growth and BRAF-inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2515.

Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be [...] Research Article Hematology

Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

BACKGROUND:Sweet's syndrome (SS) is a febrile neutrophilic dermatosis that has been clinically linked to hematological malignancies, particularly myelodysplastic syndrome (MDS), in a number of case series. Many epigenetic changes underlying MDS have been identified, such as a mutation in the isocitrate dehydrogenase 1 (IDH1) gene, which causes DNA hypermethylation and alteration of a number of genes that lead to leukemogenesis. However, the pathogenesis of malignancy-associated SS is unknown.CASE REPORT:We present two patients who were diagnosed with SS and concomitant IDH1-mutated MDS. Immunohistochemical staining of their skin lesions showed neutrophils diffusely positive for the IDH1 mutation.CONCLUSION:These cases demonstrate that IDH1 mutation may be implicated in the pathogenesis of malignancy-associated SS. Future investigation to elucidate this pathway is warranted. Establishing this molecular link can provide an earlier identification of patients with SS who are also at increased risk for developing MDS.

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Abstract The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow–derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine–treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98–HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846–57. ©2017 AACR.

Introduction: We described the mutational spectrum of ATLL in North American patients at ASH 2016 and found that these patients have a heterogenous mutational spectrum (abstract #1758). We also noted some significant differences in comparison to Japanese ATLL cases (Kataoka et al. 2015 PMID: 26437031). In this abstract, we determine the mutational landscape of larger cohort of 27 cases and report on the presence of distinct variants in epigenetic and histone modifying genes.

Candida tropicalis is a pathogenic yeast with worldwide recognition as the second or third more frequently isolated species in Latin America, for both superficial and systemic infections. Because of its high prevalence, and growing clinical interest, it is essential to understand genetic variability patterns of this important Candida species in the tropics. Besides belonging to the human normal microbiota, C. tropicalis may be found in other warm blood animals and in the environment, including water and sand of beaches. The aims of the present study were to evaluate genotypic and phenotypic variability of 62 isolates of C. tropicalis obtained from the coastal environment in Northeast Brazil using microsatellite and MALDI-TOF/MS comparisons. There was a relatively low correspondence between these typing techniques employed. Therefore, further studies are needed to consolidate the use of MALDI-TOF/MS as a yeast typing tool. Nevertheless, the two methods employed demonstrated the heterogeneity of C. tropicalis in a coastal environment. We also found relative maintenance of the population structure within the same season, which may reinforce the idea that this species presents the potential to remain in the environment for a long period of time. In addition, highly related strains were found within different geographic points of collection, demonstrating that this strain may be dispersed at long distances, probably influenced by anthropogenic actions and driven by the sea tides and wind.

Tushar D. Bhagat, Si Chen, Matthias Bartenstein, A.,Trevor Barlowe, Dagny Von Ahrens, Gaurav S. Choudhary, Patrick Tivnan, Elianna Amin, Mario Marcondes, Mathijs A. Sanders, Remco M. Hoogenboezem, Suman Kambhampati, Nandini Ramachandra, , Iaonnis Mantzaris, Vineeth Sukrithan, Remi Laurence, Robert Lopez, Prafullla Bhagat, Orsi Giricz, Davendra Sohal, Amittha Wickrema, Cecilia Yeung, Kira Gritsman, Peter Aplan, Konrad Hochedlinger, Yiting Yu, Kith Pradhan, Jinghang Zhang, John M. Greally, Siddhartha Mukherjee, Andrea Pellagatti, Jacqueline Boultwood, Britta Will, Ulrich Steidl, Marc H.G.P. Raaijmakers, H. Joachim Deeg, Michael G. Kharas*, Amit Verma

Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

1Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. 2Department of Hematology, Tianjin Medical University General Hospital, Tianjin, China. 3Department of Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA. 4Department of Pathology, Peggy and Stephenson Cancer Center, Oklahoma University Health Sciences Center, Oklahoma City, Oklahoma, USA. 5Department of Biotherapy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Immunology and Biotherapy, Key Laboratory of Cancer Prevention and Therapy, Tianjin, PR China. 6Translational Science, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA. 7Department of Hematopathology and Laboratory Medicine, 8Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA. 9Leukemia Center, Memorial Sloan Kettering Cancer Center, New York City, New York, USA.

Abstract Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls. In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis. Citation Format: Matthias Bartenstein, Lanzhu Yue, Wanke Zhao, Wanting Tina Ho, Cem Murdun, Adam W. Mailloux, Ling Zhang, Anjali Budhakoti, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit K. Verma, Pearlie K. Epling-Burnette. TGF-ß signaling inhibition counteracts myelofibrosis in MPN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2017-954