ALBERT

Description: Although the high mobility group A1 (HMGA1) oncogene is widely overexpressed in high-risk hematopoietic malignancies and other aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. We showed that HMGA1 induces leukemic transformation in cultured human lymphoid cells. Inhibiting HMGA1 expression blocks the transformed phenotype in cultured human leukemia and lymphoma cells. We also engineered HMGA1a transgenic mice and all mice develop aggressive lymphoid malignancy which closely models human T-cell acute lymphoblastic leukemia. Because HMGA1 participates in transcriptional regulation, we hypothesize that it drives leukemic transformation by dysregulating specific molecular pathways. To discover genes targeted by HMGA1 in leukemic transformation, we performed gene expression profile analysis. The signaltransducer andactivator oftranscription 3 (STAT3) gene was identified as a critical downstream target of HMGA1. STAT3 mRNA and protein are up-regulated in leukemic cells overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a. HMGA1a binds directly to a conserved region of the STAT3 promoter in vivo and activates transcription of the STAT3 promoter in human leukemia cells. Blocking STAT3 function with a small molecule, platinum compound inhibitor (CPA-7) induces apoptosis in leukemic cells from HMGA1 transgenic mice, but not in control cells. In primary, human leukemia samples, there is a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function with a dominant-negative construct in human leukemia or lymphoma cells leads to decreased cellular motility and colony formation. We also showed that treatment with a small molecule, oligonucleotide inhibitor decreases the leukemic burden in the HMGA1a transgenic mice. Our results demonstrate that the HMGA1a-STAT3 axis is a potential “Achilles heel” that could be exploited therapeutically in selected hematopoietic malignancies.

Description: Introduction: Nuclear chromatin structure is a key determinant of stem cell function and cell fate, although factors that regulate this are only beginning to emerge. While High Mobility Group A1(HMGA1) chromatin remodeling proteins are among the most abundant, nonhistone chromatin binding proteins in adult stem cells (ASCs), their role in this setting has been unknown. HMGA1/2 proteins modulate gene expression by binding to DNA, bending chromatin, and recruiting transcription factor complexes to enhancers throughout the genome. The HMGA1 gene is highly expressed during embryogenesis with low or undetectable levels in mature, differentiated tissues. In cancer, HMGA1 re-expression occurs through oncogenic transcription factors, other epigenetic alterations, or in rare cases, chromosomal translocation events. Importantly, HMGA1 levels correlate with adverse clinical outcomes in diverse malignancies. We previously reported that Hmga1 transgenic mice develop leukemic transformation by inducing transcriptional networks involved in stem cell function and cell cycle progression. Methods: To elucidate the role of Hmga1 in normal development and ASCs in vivo, we generated mouse models with transgenic overexpression or deletion of Hmga1. To define the function of Hmga1 in adult stem cells (ASCs), we used gain-of-function (overexpression) and loss-of-function (silencing or genetic deletion) approaches in human and murine intestinal stem cells (ISCs) and hematopoietic stem and progenitor cells. Results:Transgenic mice overexpressing Hmga1 in ISCs develop hyperproliferation, aberrant crypt formation, and polyposis in the intestinal epithelium by expanding the ISC and niche compartments. Hmga1 enhances self-renewal in ISCs by amplifying Wnt/β-catenin signaling, inducing genes that encode both Wnt agonist receptors and downstream Wnt effectors. Surprisingly, Hmga1 also "builds" an epithelial niche by directly up-regulating Sox9 to induce Paneth cell differentiation. Paneth cells constitute the epithelial ISC niche by secreting Wnt agonists. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestine, HMGA1 and SOX9 are highly correlated, and both become up-regulated in colorectal cancer. Human CD34+ cells engineered to overexpress Hmga1 expand more efficiently, while those with Hmga1 deficiency have defective proliferation and colony forming capability. Both colony number and size were decreased, and differentiation was skewed towards myeloid lineages. In mice, Hmga1 deletion causes partial embryonic lethality; over 50% of expected offspring die before mid-gestation. Those that survive develop premature aging phenotypes with early kyphosis, decreased bone density, grip strength, gait velocity, and hearing deficits. Knock-out mice also have early thymic aplasia, decreased numbers of early T-cell precursors, as well as decreased B-cell differentiation. Long-term (LT)-hematopoietic stem cells were decreased and preliminary data suggests aberrant regenerative function in serial, competitive transplant experiments.Preliminary ChIP-seq and gene expression studies in CD34+ cells suggest that Hmga1 regulates transcriptional networks involved in Wnt, JAK-STAT, and PI3K signaling. Conclusions:Our results in ASCs reveal a novel role for Hmga1 in tissue homeostasis by inducing pathways involved in Wnt and regenerative function. In ISCs, Hmga1 maintains both the stem cell pool and niche compartment whereas deregulated Hmga1 may perturb this equilibrium during carcinogenesis. Functional studies in HSCs suggest that Hmga1 also regulates self-renewal, regenerative potential, and the capacity for balanced differentiation. These findings indicate that HMGA1 is required for normal stem cell function, both during embryogenesis, and postnatally, in ASCs. Our prior work in tumor models demonstrates that a subset of HMGA1 stem cell pathways are hi-jacked by cancer cells to drive tumor progression. Together, these studies provide compelling rationale for further research to determine how to harness HMGA1 for regenerative medicine and to target it in cancer therapy. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2323 Although recent studies have identified genes important in hematopoietic stem cells (HSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), the molecular underpinnings of normal stem cell function are unclear. A better understanding of key stem cell pathways will be essential for the safe use of stem cells in regenerative medicine and should also uncover novel therapeutic targets in aggressive hematologic malignancies and other stem-like cancer cells. To elucidate the molecular underpinnings of “stemness”, we investigated transcriptional networks in pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. These proteins bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. HMGA1 is highly expressed during embryogenesis with low or undetectable levels in adult, differentiated tissues. HMGA1 is also enriched in HSCs, hESCs, iPSCs, refractory leukemia, and poorly differentiated solid tumors. Our group discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive leukemia in transgenic mice. We also found that high levels of HMGA1 expression correlate with relapse in acute lymphoblastic leukemia. Together, these findings suggest that HMGA1 drives a stem cell phenotype during normal development, hematopoiesis, and malignant transformation. To further investigate the role of HMGA1 in a stem cell state, we compared its expression in iPSCs, hESCs, HSCs, and cancer cells. HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in HSCs and cancer cells, and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation in hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells (mesenchymal stem cells, HSCs, and fetal lung fibroblasts) to an iPSC together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC or OSKM). HMGA1 results in an increase in the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign, teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In summary, our findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in hematologic and other malignancies or exploited in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1371 Although the high mobility group AT-hook 1 (HMGA1) gene functions as a potent oncogene in experimental models and high expression of HMGA1 portends a poor prognosis in diverse tumors, its role in leukemogenesis has remained elusive. We showed previously that HMGA1 induces leukemic transformation in cultured cells and causes aggressive lymphoid leukemia in transgenic mice. Inhibiting HMGA1 expression blocks colony formation in human lymphoid leukemia cells in vitro. Moreover, high levels correlate with relapse in childhood acute lymphoblastic leukemia (ALL), suggesting that it plays an important role in ALL. Because HMGA1 functions as a chromatin remodeling protein that modulates gene expression, we hypothesized that it drives leukemogenesis by dysregulating specific genes and pathways. To identify genes and cellular pathways induced by HMGA1 that could be targeted in therapy, we performed global gene expression profile analysis from lymphoid cells from the HMGA1 transgenic mice at different stages in tumorigenesis. All HMGA1 transgenics succumb to lymphoid malignancy with complete penetrance by 8–12 months. Pooled RNA samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) were analyzed for differential expression of 〉20,000 unique genes by microarray analysis (Affymetrix) using both a parametric and nonparametric approach. A subset of differentially expressed genes was confirmed using quantitative, RT-PCR. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis (IPA; www.ingenuity.com) and Gene Set Enrichment Analysis. To determine if these genes and pathways were relevant in human ALL, we knocked down HMGA1 expression in human ALL cells and assessed expression of a subset of the differentially expressed genes. Early in leukemogenesis (at 2 months), 113 genes were differentially expressed in the HMGA1 transgenics compared to controls. In established leukemia (12 months), 715 genes were differentially expressed. In established tumors, the dysregulated genes are involved in cancer, cell cycle regulation, and cell-mediated immune response by Ingenuity Pathway Analysis. Geneset enrichment showed that embryonic stem cell genes are enriched in the established leukemic cells. At both early and late stages in leukemogenesis, differentially regulated genes are involved in cellular development, hematopoiesis, and hematologic development. Early in leukemogenesis, most of the significantly dysregulated genes are involved in the inflammatory response and included NF-kappaB as a major node. In human ALL cells, knock-down of HMGA1 also resulted in knock-down of genes identified in our transgenic model, suggesting that these HMGA1 regulated genes are also relevant to human ALL. In summary, we found that HMGA1 induces inflammatory pathways early in leukemogenesis and pathways involved in embryonic stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dysregulates genes involved in cellular development and hematopoiesis at both early and late stages of tumorigenesis. Some of these HMGA1 pathways were also present in human ALL cells. Moreover, these results provide mechanistic insight into HMGA1 function at different stages in leukemogenesis and point to cellular pathways that could serve as therapeutic targets in ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Background Although the high mobility group A1 (HMGA1) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. HMGA1 functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, HMGA1 is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from HMGA1a transgenic mice at different stages in tumorigenesis. Results RNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of 〉 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors. Conclusions We found that HMGA1 induces inflammatory pathways early in lymphoid tumorigenesis and pathways involved in stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dyregulates genes and pathways involved in stem cells, cellular development and hematopoiesis at both early and late stages of tumorigenesis. These results provide insight into HMGA1 function during tumor development and point to cellular pathways that could serve as therapeutic targets in lymphoid and other human cancers with aberrant HMGA1 expression.