Search

RNA-binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be dysregulated in hematological malignancies. Here we identify the RBP RNA-binding motif protein, X-linked (RBMX; also known as hnRNPG), and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX and RBMXL1 were overexpressed in individuals with acute myeloid leukemia (AML) compared to healthy individuals, and RBMX/RBMXL1 loss delayed leukemia development. RBMX/RBMXL1 loss lead to global changes in chromatin accessibility as well as chromosomal breaks and gaps. We found that RBMX and RBMXL1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (also known as heterochromatin protein 1 alpha (HP1-α)), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/RBMXL1 depletion effects on cell growth and apoptosis. Overall, we determined that RBMX and RBMXL1 control leukemia cell survival by regulating chromatin state through the downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX and RBMXL1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.

Islet autotransplantation (IAT) is increasingly being performed to mitigate against the diabetic complications of pancreatic resection in patients with benign inflammatory pancreatic disorders; however, the glycemic benefit of IAT in patients undergoing partial pancreatic resection is not known. We aimed to determine whether IAT improved glycemic outcomes in patients undergoing distal pancreatectomy for benign inflammatory disease. We performed a multicenter, retrospective case-control study of patients who underwent distal pancreatic resection with IAT at two U S tertiary care centers. The primary outcome was the mean change in pre- vs post-operative HgA1c following transplant as well as the development of new post-operative diabetes. Nine patients requiring distal pancreatectomy for benign disease underwent IAT and were compared to 13 historical controls without IAT. Baseline characteristics were similar between groups. With a median follow-up of 22 months, those who received an IAT had a smaller increase in their pre- vs post-operative HgA1c (0.42 vs 2.83, P = .004), and one case patient (14.3%) vs three control patients (23.1%) developed new post-operative diabetes (P = .581). We conclude that patients undergoing distal pancreatic resection for benign inflammatory disease should be considered for IAT, as long-term glycemic outcomes appear to be improved in those undergoing transplant.

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease., Several studies have recently demonstrated the role of the MSI2 RNA binding protein in normal and malignant haematopoietc stem cells. In this study, the authors show that MSI2 is required for maintaining myelodysplastic syndrome stem cells in mice and that MSI2 expression predicts poor prognosis in patients affected by this disease.

SummaryMembers of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

Hematopoiesis is maintained throughout life by self-renewing hematopoietic stem cells (HSCs) that differentiate to produce both myeloid and lymphoid cells. The NR4A family of orphan nuclear receptors, which regulates cell fate in many tissues, appears to play a key role in HSC proliferation and differentiation. Using a NR4A1GFP BAC transgenic reporter mouse we have investigated NR4A1 expression and its regulation in early hematopoiesis. We show that NR4A1 is most highly expressed in a subset of Lin−Sca-1+c-Kit+ CD48−CD150+ long-term (LT) HSCs, and its expression is tightly associated with HSC quiescence. We also show that NR4A1 expression in HSCs is induced by PGE2, a known enhancer of stem cell engraftment potential. Finally, we find that both NR4A1GFP+ and NR4A1GFP− HSCs successfully engraft primary and secondary irradiated hosts; however, NR4A1GFP+ HSCs are distinctly myeloid-biased. These results show that NR4A1 expression identifies a highly quiescent and distinct population of myeloid-biased LT-HSCs. Stem Cells 2015;33:278–288

Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia.

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known. Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis. To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance. To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA. To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines. In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. Disclosures No relevant conflicts of interest to declare.

+ The first four authors contributed equally to this project. Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. LSCs are characterized by their gain of a self-renewal program that is normally associated with hematopoietic stem cells (HSCs). Previously we have shown that the RNA binding protein, Msi2 contributes to both HSC and myeloid leukemia function. Elevated MSI2 expression predicts a poor prognosis in a variety of leukemias and shRNA-mediated depletion in human AML cell lines reduces proliferation, increases differentiation and induces apoptosis. Despite these in vitroand correlative studies, MSI2’s molecular mechanism is not known and its role in LSC function has not been assessed. To elucidate MSI2’s role in LSC function, we utilized the MLL-AF9 leukemia mouse model. Initially we found MSI2 was elevated in the LSC enriched compartment (c-KitHigh cells) compared to non-LSCs (c-KitLow cells) based on flow cytometric intracellular staining. Therefore, to establish a model to study Msi2 and its contribution to myeloid LSCs, we have utilized the Msi2 conditional knockout mice that we previously crossed (Msi2f/f) into an Mx1-Cre background to generate the Msi2Δ/Δallele (injection of polyinositol-polycytosine; pIpC). In order to test if Msi2 is critical for MLL-AF9 mediated initiation, we transduced control Msi2f/f and Msi2Δ/ΔLin- Sca1+ c-Kit+cells (LSKs) with MLL-AF9 expressing retroviruses co-expressing GFP. Msi2 deleted LSKs or granulocyte-monocyte progenitors (GMPs) transduced with MLL-AF9 demonstrated delayed leukemogenesis with dramatically reduced diseased burden. Msi2 deficient leukemias were found to have a 4-fold reduced phenotypic LSC population and were more differentiated based on cellular morphology. Msi2 deficient leukemias failed to transplant into secondary recipients demonstrating that Msi2 is required for maintaining LSCs. Deletion of Msi2after leukemia engraftment led to a delay in leukemogenesis indicating that Msi2 is also important for leukemia maintenance. Gene expression profiling of the Msi2 ablated LSCs resulted in a loss of the HSC/LSC program and an increase in differentiation gene sets. The gene signature from the Msi2 deleted murine LSCs (121 genes) was overlapped and subjected to unsupervised clustering with the gene expression profiles from 336 AML patients (ECOG1900 dataset). This analysis resulted in distinct clusters that had differential MSI2 expression and the MSI2“high” cluster predicted a worse clinical outcome when compared to the other clusters. Overlapping of the differential transcriptional analysis of the Msi2 deleted murine LSCs with our global MSI2 direct mRNA targets (HITS-CLIP) led us to identify that MSI2 binds to transcripts that are associated with the downstream MLL self-renewal program, including Myc and Ikzf2. Ikzf2 is a member of the Ikaros transcription factor family and is known to regulate lymphocyte development by controlling regulatory T-cell function and chromatin remodeling. Ikzf2 shRNA mediated depletion resulted in reduced colony formation, decreased proliferation and increased apoptosis. The MLL associated targets were also reduced, which included Bcl-2 and Hoxa9. In contrast to its tumor suppressor role in hypodiploid B-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell maintenance. Thus, we provide evidence that MSI2 maintains the oncogenic LSC epigenetic program and the rationale for clinically targeting MSI2 in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.