ALBERT

Description: Introduction The bone marrow microenvironment regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), through a network dependent on cell-cell interaction. This interaction is mediated by morphogens, the extracellular matrix and cell adhesion molecules expressed and secreted by various cell types in the HSPC niche. Mesenchymal stromal cells (MSCs), as the major cellular component, maintain the stemness properties of the niche. The microenvironment thus becomes conducive for HSPCs to remain quiescent, thereby enabling long term self-renewal. Therefore, the safe haven in the bone marrow microenvironment and its constituent cell types can be targeted during tumorigenesis, thus making the niche neoplastic. Dissemination of breast cancer cells into the bone marrow has been described even in the early stages of the disease. The present study focuses on the influence of breast carcinomas on the genetic and functional profile of mesenchymal and hematopoietic progenitor cells of the bone marrow niche. Methods In vitro coculture models of breast cancer cell lines- MDA-MB231, MCF-7 and primary MSCs derived from the bone marrow of healthy donors were used in the study. Atomic- force microscopy based single-cell force spectroscopy (AFM-SCFS) and fluorescence based assays were used for cell adhesion experiments. Hydrogel based culture systems were used for 3-dimensional cocultures of breast cancer cells and MSCs. Hypoxic and normoxic culture conditions (0.5% and 20% oxygen respectively) were used for the experiments. Results The breast cancer cell lines caused a significant reduction in HSPC adhesion to MSCs (88% by MDA-MB 231 cells; p

Description: Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. Here we characterize the immunological features of the malignant clone and alterations in the immune microenvironment in TP53 mutant and wild type MDS and sAML patients. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of TP53 mutant patients, which is associated with MYC upregulation and marked down-regulation of MYC's negative regulator miR-34a, a p53 transcription target. Notably, TP53 mutant patients display significantly reduced numbers of bone marrow infiltrating OX40+ cytotoxic T-cells and helper T-cells, as well as decreased ICOS+ and 4-1BB+ NK cells. Further, highly immunosuppressive regulatory T-cells (i.e., ICOSHigh/PD-1neg) and MDSCs (PD-1low) are expanded in TP53 mutant cases. Finally, a higher proportion of bone marrow infiltrating ICOSHigh/PD-1neg Tregs is a highly significant independent predictor of overall survival. We conclude the microenvironment of TP53 mutant MDS and sAML has an immune privileged, evasive phenotype that may be a primary driver of poor outcomes, and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly-defined subpopulation.

Description: Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Incyte Corporation: Research Funding.