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Supp Table 1: MDS Patient Disease Characteristics Supp Table 2: Pathways affected by hypermethylation in MDS stroma Supp Table 3: Genes that are methylated and underexpressed in MDS stroma Supp Table 4. MDS patient characteristics for primary MSC sample isolation Supp table 5: WNT Targets

Supplementary Methods

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.

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

Intrahepatic cholangiocarcinoma (ICC) is the second-most common primary liver malignancy after hepatocellular carcinoma. While surgical resection with negative margin is the only curative treatment, ICC has very high rate of recurrence, up to 60-70% after curative resection. We reviewed the current data available on risk factors for ICC recurrence, recurrence pattern (location and timing), treatment options, and future directions. The risk factors for recurrence include elevated preoperative CA19-9, presence of liver cirrhosis, nodal metastasis, positive margins, and vascular invasion. Understanding different recurrence patterns, timing course, and risk factors for early recurrence is important to tailor postoperative surveillance and select treatment strategies including systemic or locoregional therapy. Re-resection can be considered for a selected patient population at experienced centers, and can yield long-term survival. ICC remains a dismal disease given the high likelihood of recurrence. Advances in our understanding of the genomic landscape of ICC are beginning to identify targetable alterations in ICC in subsets of patients that allow for personalized treatment.

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.

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.

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Living donor liver transplant (LDLT) has markedly reduced waiting list mortality and expanded access to life-saving therapy for thousands across the globe. Application of minimally invasive surgery to donor hepatectomy has been markedly slower than in many other aspects of surgery. For donors undergoing hepatectomy, many long-lasting issues related to donation appears to involve the large incision typically used for donor hepatectomy. This has provided significant motivation for a minimally invasive approach to donor hepatectomy. Pure laparoscopic donor hepatectomy was initially reported in 2002 but adoption has been slowed by concerns about hemostasis, donor safety and recipient outcomes. Highly select centers have successfully developed minimally invasive living donor programs and have been reporting outcomes and techniques utilized. We review the technical approaches have been learned by early adopters. We discuss some of the pitfalls and challenges associated with laparoscopic donor hepatectomy. We discuss the reported experiences with the learning curve for this complex operation. We discuss the future of minimally invasive surgery for donor hepatectomy.

Alternative RNA splicing and mutations in spliceosome genes are common features of MDS and AML, however which misspliced genes contribute the malignant state remains unclear. A global analysis of intron and exon usage in AML revealed enrichment of alternatively spliced genes associated with inflammatory and immune pathways in leukemic cells as compared to normal cells. The gene with the greatest differential isoform expression was IRAK4, a serine/threonine kinase downstream of toll-like receptor (TLR) signaling. Examination of IRAK4 by RNA sequencing showed that normal cells preferentially express an IRAK4 isoform resulting from exclusion of the exon 4, which encodes a protein lacking the N-terminal death domain (IRAK4-Short). In contrast, a large proportion of MDS/AML samples show increased expression of an IRAK4 isoform that retains exon 4, encoding the full-length protein (IRAK4-Long). Of all somatic genetic mutations associated with AML, mutation of U2AF1 (S34F) significantly correlated with inclusion of exon 4 and expression of IRAK4-Long. Examination of IRAK4 exon 4 usage in CD34+ cells from genetically-defined MDS patient samples revealed that nearly all MDS patient samples containing mutations in U2AF1 exhibited increased inclusion of exon 4 as compared to WT U2AF1 MDS samples or healthy controls. Strikingly, SF3B1 mutant MDS patient samples also exhibited increased expression of IRAK4-Long, suggesting a unifying mechanism of IRAK4 isoform expression in spliceosome mutant MDS/AML. Higher expression of IRAK4-Long in MDS CD34+ cells or AML blasts is associated with poor prognosis, and correlates with elevated blast counts and transfusion dependency in MDS. Utilizing a splicing reporter containing exon 4 and flanking intron sequences of IRAK4, U2AF1-S34F induced retention of the cassette exon 4, while WT U2AF1 mediated exclusion of exon 4. Ectopic expression of U2AF1-S34F in AML cells resulted in significant retention of IRAK4 exon 4 and expression of IRAK4-Long protein. Functional characterization of the IRAK4 isoforms in human AML cell lines revealed that IRAK4-Long induces NF-kB activation, suggesting that mutations in U2AF1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Importantly, U2AF1-S34F AML cells were more sensitive to pharmacologic inhibition of IRAK1/4 as compared to isogenic cells with WT U2AF1. CA-4948, a potent oral small-molecule inhibitor of IRAK4, blocked IRAK4-mediated signaling in TLR-stimulated THP1 AML cells, and decreased leukemic burden in xenografted mice. Finally, CA-4948 treatment led to reduced leukemic engraftment of primary patient MDS/AML samples with expression of IRAK4-Long. Taken together, we find that mutations in U2AF1 and SF3B1 induce expression oftherapeutically targetable"active" IRAK4 isoforms and provide a genetic link to chronic innate immune signaling and IRAK1/4 activation in MDS and AML. Disclosures Steidl: Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding. Booher: Curis, Inc: Employment, Equity Ownership.

Myelodysplastic syndrome (MDS) & Acute Myeloid Leukemia (AML) are hematologic malignancies that arise from a population of aberrant hematopoietic stem cells (HSCs). Overactivated innate immune signaling pathways such as IRAK1, TRAF6, IL1RAP, S100A9 and IL8 have been demonstrated in MDS/AML and play important roles in propagation of disease. IRAK4 (interleukin-1 receptor-associated kinase 4), is a protein kinase involved in signaling innate immune responses and forms a critical signaling complex with IRAK1. To determine its role in disease pathobiology, we analyzed transcriptomic data from CD34+ stem and progenitor cells from 183 MDS patients and found significantly increased expression of IRAK4 in MDS samples belonging to the high risk RAEB category (Refractory anemia with excess of blasts, N=80, P Value To functionally determine the role of IRAK4 in MDS/AML pathogenesis, we utilized CA-4948, a potent, oral, small-molecule inhibitor of IRAK4, to assess the effect of inhibiting IRAK4 catalytic activity. In vitro, CA-4948 blocked downstream NF-κB pathway signaling, including secretion of proinflammatory cytokines, in Toll-like receptor stimulated THP1 leukemic cells. CA-4948 was tested in clonogenic assays from primary MDS and AML samples. MDS and AML are associated with block in differentiation that leads to cytopenias that are the cause of morbidity in these patients. Treatment with CA-4948 led to increased erythroid and myeloid differentiation in a majority of samples. Furthermore, drug treatment led to decreased viability of MDS/AML stem cells (CD34+/CD38-/Lin-ve) In vivo studies using a THP1 leukemia xenograft model in NSG mice demonstrated that CA-4948 was well tolerated and led to significantly decreased disease burden after 6 weeks of treatment. In conclusion, we demonstrate that IRAK4 is upregulated in stem and progenitor cells in MDS and AML and is an adverse prognostic marker. Importantly, a novel, specific, inhibitor of IRAK4 shows preclinical in vitro and in vivo efficacy in MDS and AML models. Citation Format: Gaurav S. Choudhary, Tushar D. Bhagat, Maria Elena S. Samson, Shanisha Gordon, Dagny Von Ahrens, Kith Pradhan, Aditi Shastri, Andrea Pellagatti, Jacqueline Boutlwood, Robert N. Booher, Ulrich Steidl, Amit Verma. Efficacy of novel IRAK4 inhibitor CA4948 in AML and MDS [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 127. doi:10.1158/1538-7445.AM2017-127