ALBERT

Description: Abstract 555 The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineage of the bone marrow, resulting in peripheral cytopenias and the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). The molecular and cellular events involved in MDS development are yet to be elucidated. Large scale cytogenetic aberrations such as del(5q) have been associated with many types of MDS. Previous work from our lab has shown that the chromosomal band 5q32–33 harbours microRNAs (miR-145 and miR-146a), haploinsufficiency of which recapitulates the megakaryocytic dysplasia and neutropenia associated with del(5q) MDS. Ingenuity pathway analysis predicts that innate immune signaling is one of the most highly deregulated pathways by loss of these two miRNAs. Two innate immune signaling molecules, TNF receptor associated factor 6 (TRAF6) and Toll/interleukin-1 receptor associated protein (TIRAP), are targets ofmiR-146a and miR-145 respectively. While the role of TRAF6 in del(5q) MDS has been investigated, little is known about the role of TIRAP in MDS pathogenesis. To investigate the role of TIRAP in aberrant hematopoiesis, we transplanted lethally irradiated mice with bone marrow cells expressing TIRAP or vector control. TIRAP transplanted mice develop an MDS-like bone marrow failure characterized by anemia, neutropenia, and thrombocytopenia as early as 4 weeks post-transplant (unlike TRAF6 transplanted mice which succumb to BMF or AML approximately 4 months post-transplant). The discrepancy between TIRAP and TRAF6 disease progression models suggests that TRAF6 independent signaling originating from TIRAP may be responsible for the rapid bone marrow failure onset. Furthermore, TIRAP transplanted mice display hypocellular marrows characterized by increased apoptosis as measured by Annexin V/PI staining, similar to low risk MDS patients. In MDS, normal hematopoiesis is blocked in the non-disease fraction of bone marrow by autoimmunity associated cytokines. To identify factors that may be responsible for suppression of normal hematopoiesis in our BMF model, we performed cytokine expression profiling of TIRAP expressing marrow. Quantitative RT-PCR showed increased expression of both IL-10 and IFNγ in TIRAP expressing marrow compared to vector control. Interestingly, IFNγ is known to suppress hematopoiesis has been shown to be overexpressed in BM mononuclear cells in MDS and other BMF conditions. Also, IL-10 levels have been shown to be elevated in high risk MDS patients. To investigate the role of IL-10 and IFNγ in TIRAP mediated marrow failure, we transduced IL-10 −/− and IFNγ −/− marrow with TIRAP or vector control, and transplanted wildtype leathally irradiated mice. Loss of IFNγ but not IL-10 partially rescues the pancytopenia phenotype and prevents early death due to bone marrow failure. Interestingly however, mice transplanted with TIRAP expressing IFNγ −/− bone marrow succumb to a myeloproliferative disorder at later time points, suggesting that the factors responsible for myelosuppression are the same ones responsible for progression to AML. Disclosures: No relevant conflicts of interest to declare.

Description: The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment results in dysplasia of the marrow cells and ineffective hematopoiesis. As a result, the primary cause of mortality in these patients is eventual bone marrow failure although MDS patients also have a significantly increased risk of transformation to acute myeloid leukemia (AML). The most common karyotypic change in MDS is an interstitial deletion of the long arm of chromosome 5, del(5q) MDS. Patients with an isolated interstitial deletion of chromosome 5q are referred to as having 5q- syndrome. Mapping of the commonly deleted region (CDR) within 5q- syndrome has identified a 1.5-megabase region on band 5q32. MicroRNA (miRNA) -143 and -145 are located within the CDR of del(5q) MDS and have been implicated in the pathogenesis of the disease. However, their functional role in myelodysplastic syndromes has not been well studied. To investigate the role of miR-143 and miR-145, we utilized a gene-targeted mouse model containing deletion of miR-143 and miR-145. Here we show that mouse marrow lacking miR-143 and miR-145 have a decrease in short-term repopulating HSC and progenitors of the myeloid lineage by flow cytometry, as well as of hematopoietic progenitor activity using colony forming assays. Additionally, we performed a limiting dilution assay of miR-143-/-145-/- bone marrow and observed significantly fewer functional HSCs compared to wildtype marrow. To explore the molecular mechanism behind this defect, we performed Ingenuity Pathway Analysis of the predicted targets of miR-143 and miR-145. We identified the transforming growth factor-beta (TGFβ)-signaling pathway as a common target of these two miRNAs. Gene Set Enrichment Analysis of del(5q) using mRNA expression of MDS patient CD34+ marrow cells show an enriched TGFβ-signature compared to healthy controls. In addition, the defect in hematopoietic progenitor activity in miR-143-/-145-/- marrow can be rescued by inhibiting Smad3 using the chemical inhibitor SIS3. We validated the TGFβ pathway adaptor protein, Disabled-2 (DAB2), as a target of miR-145 and show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of DAB2. Enforced expression of DAB2 in mouse marrow is able to recapitulate many of the features of miR-143-/-145-/- mice. DAB2 overexpressing marrow formed significantly fewer colonies in progenitor assays, and in competitive transplants, vector-transduced marrow was able to out compete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. Interestingly, compared to wildtype mice, aged miR-143-/-145-/- mice showed decreased hemoglobin and platelet counts with elevated white blood cell counts. This phenotype was also observed in a subset of mice with enforced DAB2 expression where a proportion of mice developed a transplantable myeloproliferative disorder. Together, our data identifies a role for miR-143 and miR-145 in the pathogenesis of del(5q) MDS where their loss results in a defect in HSC activity. We observe that the TGFβ signaling pathway is activated in patient marrow and we validate DAB2 as a direct target of miR-145. We provide evidence that the defect observed in miR-143-/-145-/- marrow is mediated in part by DAB2 where its enforced expression leads to a defect in HSC self-renewal but contributes to myeloproliferation. Disclosures Karsan: Celgene: Research Funding.