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Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs act as a barrier to therapy and release signals promoting tumor growth and invasion, the stroma restricts tumor growth in mouse models. The High Mobility Group A1 (HMGA1) protein is an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. Although HMGA1 is highly expressed in embryonic and adult stem cells, it is silenced postnatally in most differentiated cells. HMGA1 becomes aberrantly re-expressed in diverse tumors where high levels predict adverse clinical outcomes. In PDAC, HMGA1 is detected only in late-stage precursor lesions or invasive tumors, but not in normal pancreas nor in early precursor lesions. Moreover, high HMGA1 nuclear staining associates with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program whereby HMGA1 recruits stromal CAFs and drives tumor progression by inducing FGF19. Silencing HMGA1 in PDAC cell lines slows proliferation, disrupts oncogenic properties in vitro (migration, invasion, clonogenicity, 3D sphere formation), and depletes tumor initiator cells in xenograft assays. RNA sequencing revealed transcriptional networks up-regulated by HMGA1 that function in cell signaling and proliferation; we focused on FGF19 as a potential mediator of tumor-stromal crosstalk. HMGA1 binds directly to the FGF19 promoter and recruits active histone marks (H3K4me2, H3K27Aac) to induce gene expression and protein secretion from PDAC cells. Silencing FGF19 recapitulates effects of HMGA1 silencing, disrupting oncogenic and cancer stem cell properties in vitro while depleting tumor initiator cells in vivo. In co-culture experiments, FGF19 is required for CAF migration across a membrane towards PDAC cells. Silencing HMGA1, FGF19, or treatment with FGF19 inhibitors disrupts CAF recruitment. Silencing HMGA1 or FGF19 in PDAC cells also decreases both the desmoplastic stroma and tumor growth in mouse xenografts. In primary tumors, co-expression of HMGA1 and FGF19 predict decreased survival. Together, our results reveal a novel paradigm whereby tumor cells collaborate with CAFs via HMGA1 and FGF19 to drive progression, thus illuminating FGF19 as a rational therapeutic target for PDACs overexpressing HMGA1 and FGF19. Citation Format: Lionel Chia, Shuai Shuai, Jung-Hyun Kim, Woo Jung Sung, Ruitao Zhang, Tait Huso, Leslie Cope, Karen Reddy, Linda M. Resar. HMGA1 induces FGF19 to drive tumor progression and recruit cancer associated fibroblasts in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2414.

Background & Objectives: Chromatin structure is the single most important feature that distinguishes a cancer cell from a normal cell histologically. Chromatin remodeling proteins regulate chromatin structure and high mobility group A (HMGA1) proteins are among the most abundant, nonhistone chromatin remodeling proteins found in cancer cells. These proteins include HMGA1a/HMGA1b isoforms, which result from alternatively spliced mRNA. The HMGA1 gene is overexpressed in cancer and high levels portend a poor prognosis in diverse tumors. HMGA1 is also highly expressed during embryogenesis and postnatally in adult stem cells. Overexpression of HMGA1 drives neoplastic transformation in cultured cells, while inhibiting HMGA1 blocks oncogenic and cancer stem cell properties. Hmga1 transgenic mice succumb to aggressive tumors, demonstrating that dysregulated expression of HMGA1 causes cancer in vivo. HMGA1 is also required for reprogramming somatic cells into induced pluripotent stem cells. HMGA1 proteins function as ancillary transcription factors that bend chromatin and recruit other transcription factors to DNA. They induce oncogenic transformation by activating or repressing specific genes involved in this process and an HMGA1 “transcriptome” is emerging. Although prior studies reveal potent oncogenic properties of HMGA1, we are only beginning to understand the molecular mechanisms through which HMGA1 functions. In this review, we summarize the list of putative downstream transcriptional targets regulated by HMGA1. We also briefly discuss studies linking HMGA1 to Alzheimer’s disease and type-2 diabetes. Conclusion: Further elucidation of HMGA1 function should lead to novel therapeutic strategies for cancer and possibly for other diseases associated with aberrant HMGA1 expression.

Despite dramatic improvements in treatment and survival, acute lymphoid malignancies remain the leading cause of death in children with cancer and a highly lethal malignancy in adults.[1] Acute lym...

Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs provide a barrier to therapy and release signals that foster tumor growth and invasion, the stroma also restrains tumor growth in murine models. The High Mobility Group A1 (HMGA1) chromatin remodeling gene encodes an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. HMGA1 is also highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in most differentiated cells. In diverse, aggressive cancers, HMGA1 becomes aberrantly re-expressed where high levels portend adverse clinical outcomes. In PDAC, HMGA1 protein is detected in late stage precursor lesions and invasive tumors, but not in normal pancreas nor in early precursor lesions. Furthermore, high HMGA1 levels correlate with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program mediated by HMGA1 that recruits CAFs to drive PDAC progression. We discovered that silencing HMGA1 in multiple PDAC cell lines halts proliferation and disrupts oncogenic properties, including migration, invasion, clonogenicity, and xenograft tumorigenesis. HMGA1 silencing also impairs three-dimensional sphere formation and depletes tumor initiator cells in limiting dilution assays. Through RNA sequencing analysis comparing PDAC cells overexpressing HMGA1 to those with HMGA1 silencing, we identified FGF-19 as a potential transcriptional target of HMGA1. HMGA1 binds specifically to the FGF-19 promoter and recruits the active histone mark, H3K4me3, to activate its expression. HMGA1 is also required for FGF-19 secretion from PDAC cells. Similar to HMGA1, silencing FGF-19 blocks oncogenic and cancer stem cell properties in vitro while disrupting tumorigenesis and depleting tumor initiator cells in vivo. In co-culture experiments, HMGA1 expressed in PDAC cells amplifies FGF-19 secretion, thereby stimulating CAF migration to tumor cells across a membrane. Silencing HMGA1 or FGF-19 prevents CAF recruitment to PDAC tumor cells. Furthermore, CAF recruitment is blocked by either FGF-19 blocking antibodies or an inhibitor to the FGF-19 receptor (FGR4). In murine models, silencing HMGA1 also decreases formation of a fibroblastic stroma. Moreover, overexpression of HMGA1 together with that of FGF-19 predict decreased survival in primary human PDAC. Our results reveal a novel paradigm whereby PDAC cells collaborate with stromal CAFs via HMGA1 and FGF-19 to drive tumor progression. These data also provide insight into mechanisms for tumor progression and illuminate FGF-19 as a rational therapeutic target in PDACs with up-regulation of HMGA1 and FGF-19. Citation Format: Lionel Chia, Shuai Shuai, Lingling Xian, Jung-Hyun Kim, Ruitao Zhang, Tait Huso, David Huso, Leslie Cope, Karen Reddy, Linda Resar. HMGA1 induces FGF-19 to foster tumor-stromal cell crosstalk and drive tumor progression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 297.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to ‘build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis., The function of high mobility group A1 (Hmga1) chromatin remodelling proteins in intestinal stem cells (ISC) is unknown. Here, the authors show that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and induces Sox9 to expand the Paneth cell compartment and enrich the ISC niche.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in90% of primary pancreatic cancers, with absent or low levels in early precursor lesions.Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis.HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1.Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.

Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors for which there are no effective therapies. Emerging evidence suggests that the tumor stroma interacts with the cancer cells to induce cancer stem cell properties and drive tumor progression. In PDAC, the fibroblast stroma also provides a dense barrier preventing cytotoxic therapy from reaching PDAC cells. We previously discovered that high levels of High Mobility Group A1 (HMGA1) protein predict decreased survival in primary PDAC. Here, we report a novel role for HMGA1-FGF19 in mediating tumor-stromal interactions and tumor progression. Silencing HMGA1 in PDAC cell lines or low-passge, patient-derived cells abruptly halts proliferation. Spindle-shaped, mesenchymal cells became reprogrammed into cuboidal, more epithelial-like cells. Sensitivity to gemcitabine was enhanced and colony formation, migration, invasion, and three-dimensional (3D) sphere formation were all disrupted in cells with HMGA1 knock-down. Silencing HMGA1 also disrupted xenograft tumorigenesis and depleted cancer stem cells/tumor-initiator cells in limiting dilution tumorigenicity assays. To elucidate underlying molecular mechanisms mediating these striking phenotypes, we performed RNA-seq after silencing HMGA1 in invasive, highly metastatic, low-passage patient-derived PDAC cells (10.7). Among the genes regulated by HMGA1 were those encoding proteins involved in tumor-stromal signaling, including the fibroblast growth factor 19 (FGF19). The FGF19 gene is highly expressed in GI tumors (liver, colon, PDAC) and transgenic mice overexpressing Fgr15 (the murine homolog) in hepatocytes develop hepatocellular carcinoma (HCC). FGF19 also correlates with poor outcomes in human HCC and colon cancer, although it's role in PDAC was unknown. Here, we found that FGF19 expression is dependent upon HMGA1 in 3 different PDAC cell lines; silencing HMGA1 represses FGF19 in these cells. HMGA1 also binds directly to the FGF19 promoter at 2 predicted DNA binding sites as assessed by chromatin immunoprecipiation. To determine whether FGF19 plays a functional role in HMGA1-mediated tumor progression and cancer stem cell properties, we silenced FGF19 in PDAC cells. Similar to our results with HMGA1, silencing FGF19 impaired PDAC growth and 3D sphere formation in vitro. Because fibroblast growth factors interact with fibroblasts, we determined whether the HMGA1-FGF19 pathway was involved in tumor cell – stromal crosstalk. PDAC 10.7 cells recruit cancer-associated fibroblasts (CAFs) in a co-culture system, although this recruitment was abrogated when HMGA1 was silenced. CAF migration was also disrupted by anti-human FGF19 neutralizing antibodies. Together, these findings indicate that HMGA1 drive tumor progression and cancer stem cell properties through FGF19 and suggest that targeting the HMGA1-FGF19 pathway maybe efficaceous in PDAC. Citation Format: Shuai Shuai, Lingling Xian, Tait Huso, Karen Reddy, Linda Resar. HMGA1 drives tumor progression and recruits cancer-associated fibroblasts in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4494.

Objectives Although uterine cancer is the fourth most common cause for cancer death in women worldwide, the molecular underpinnings of tumor progression remain poorly understood. The High Mobility Group A1 ( HMGA1 ) gene is overexpressed in aggressive cancers and high levels portend adverse outcomes in diverse tumors. We previously reported that Hmga1a transgenic mice develop uterine tumors with complete penetrance. Because HMGA1 drives tumor progression by inducing Matrix Metalloproteinase ( MMP ) and other genes involved in invasion, we explored the HMGA1 - MMP - 2 pathway in uterine cancer. Methods To investigate MMP-2 in uterine tumors driven by HMGA1 , we used a genetic approach with mouse models. Next, we assessed HMGA1 and MMP-2 expression in primary human uterine tumors, including low-grade carcinomas (endometrial endometrioid) and more aggressive tumors (endometrial serous carcinomas, uterine carcinosarcomas/malignant mesodermal mixed tumors). Results Here, we report for the first time that uterine tumor growth is impaired in Hmga1a transgenic mice crossed on to an Mmp-2 deficient background. In human tumors, we discovered that HMGA1 is highest in aggressive carcinosarcomas and serous carcinomas, with lower levels in the more indolent endometrioid carcinomas. Moreover, HMGA1 and MMP-2 were positively correlated, but only in a subset of carcinosarcomas. HMGA1 also occupies the MMP-2 promoter in human carcinosarcoma cells. Conclusions Together, our studies define a novel HMGA1-MMP-2 pathway involved in a subset of human carcinosarcomas and tumor progression in murine models. Our work also suggests that targeting HMGA1 could be effective adjuvant therapy for more aggressive uterine cancers and provides compelling data for further preclinical studies.

Emerging evidence suggests that cancer cells undergo chromatin remodeling and epigenetic reprogramming to co-opt stem cell properties and drive tumor progression. The HMGA1 chromatin remodeling protein is an architectural transcription factor that binds to DNA at AT-rich sequences where it “opens” chromatin, recruits transcriptional complexes, and modulates gene expression. The HMGA1 gene is highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in differentiated tissues. HMGA1 becomes re-expressed in most high-grade cancers and high levels portend adverse clinical outcomes. In colon cancer, HMGA1 is among the genes most highly overexpressed compared to normal intestinal epithelium. We previously reported that HMGA1 drives tumor progression in colon cancer by inducing stem cell genes involved in an epithelial-mesenchymal transition. We also discovered that Hmga1 transgenic mice develop marked proliferative changes and pre-malignant polyposis in the intestinal epithelium. To determine how Hmga1 functions in the intestines during tissue homeostasis and carcinogenesis, we examined in transgenic mice and organoid models. Here, we uncover a novel role for Hmga1 in maintaining the intestinal stem cell (ISC) pool and Paneth cell niche. Hmga1 is required by ISCs to organize into three-dimensional organoids in vitro; silencing Hmga1 disrupts organoid formation and bud development. Conversely, overexpression of Hmga1 increases organoid formation, bud development, and replating efficiency, suggesting that Hmga1 enhances ISC function and/or number. We therefore crossed the Hmga1 transgenic mice onto the Lgr5-EGFP background to enumerate ISCs and found that Hmga1 expands the ISC compartment. To determine how this occurs, we performed in vivo imaging and discovered that Hmga1 enhances self-renewal of ISCs. Mechanistically, we found that Hmga1 amplifies Wnt/β-catenin signaling by inducing genes encoding both Wnt agonist receptors and downstream Wnt target genes. Surprisingly, Hmga1 also expands the Paneth cell niche, which is comprised of terminally differentiated crypt cells that secrete Wnt to support ISCs. Because Paneth cells require Sox9 for development, we determined whether Hmga1 regulates its expression. Hmga1 binds directly to the Sox9 promoter at 2 AT-rich sites to activate its expression. In human colonic epithelium, HMGA1 and SOX9 are positively correlated, and both become markedly up-regulated in colon carcinogenesis. This work not only provides new insights into the role of Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment, but also suggests that deregulated Hmga1 perturbs this equilibrium during polyposis and carcinogenesis. Our results also highlight the HMGA1-WNT-SOX9 pathway as rational therapeutic target in colon carcinogenesis. Citation Format: Lingling Xian, Dan Georgess, Li Luo, Lionel Chia, Qihua Gu, Tait Huso, Amy Belton, David Huso, Andrew Ewald, Linda M.S. Resar. HMGA1 amplifies Wnt signaling and expands the intestinal stem cell compartment to drive premalignant polyposis in transgenic mice [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 5019. doi:10.1158/1538-7445.AM2017-5019

Emerging evidence demonstrates that the high mobility group A1 (HMGA1) chromatin remodeling protein is a key molecular switch required by cancer cells for tumor progression and a poorly differentiated, stem-like state. Because the HMGA1 gene and proteins are expressed at high levels in all aggressive tumors studied to date, research is needed to determine how to 'turn off' this master regulatory switch in cancer.In this review, we describe prior studies that underscore the central role of HMGA1 in refractory cancers and we discuss approaches to target HMGA1 in cancer therapy.Given the widespread overexpression of HMGA1 in diverse, aggressive tumors, further research to develop technology to target HMGA1 holds immense promise as potent anticancer therapy. Previous work in preclinical models indicates that delivery of short hairpin RNA or interfering RNA molecules to 'switch off' HMGA1 expression dramatically impairs cancer cell growth and tumor progression. The advent of nanoparticle technology to systemically deliver DNA or RNA molecules to tumors brings this approach even closer to clinical applications, although further efforts are needed to translate these advances into therapies for cancer patients.

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.

The High Mobility Group A1 (HMGA1) gene is overexpressed in most poorly differentiated cancers and high levels portend adverse clinical outcomes, although the molecular mechanisms through which it functions are poorly understood. HMGA1 encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which modulate gene expression by bending chromatin and orchestrating the assembly of transcription factor complexes to DNA. HMGA1 is highly expressed during embryogenesis, but silenced in adult, differentiated tissues. Postnatally, HMGA1 expression is maintained in adult stem cells, such as intestinal stem cells (ISCs); however, its role in this setting has been unknown. Here, we report that Hmga1 overexpression in ISCs of transgenic mice drives expansion in the ISC compartment leading to hyperproliferation, aberrant crypt formation, and polyposis. Surprisingly, Hmga1 transgenic mice also exhibit marked expansion in terminally differentiated Paneth cells, which comprise an epithelial cell niche for ISCs. To dissect the mechanisms mediating these phenotypes, we generated three-dimensional (3D) intestinal organoids with varied expression of Hmga1. Strikingly, silencing Hmga1 in wildtype crypt cells disrupts their ability to organize into functional 3D organoids with bud formation, while crypt cells expressing ectopic Hmga1 exhibit enhanced organoid formation with increased ISC number, proliferation, and bud development. Because Wnt/β-catenin signaling is central to ISC function, we determined whether Hmga1 modulates this pathway. β-catenin protein is increased in the crypts of the Hmga1 transgenic mice and organoids. Hmga1 amplifies Wnt/β-catenin signaling by inducing both genes that encode Wnt cell surface receptors and target genes downstream of Wnt/β-catenin. Hmga1 also directly up-regulates Sox9, which is required for terminal differentiation to Paneth cells. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestinal epithelium, HMGA1 and SOX9 are highly correlated (P = 0.008), and both become up-regulated in carcinogenesis. These results reveal a novel role for Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment and suggest that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis. Citation Format: Lingling Xian, Tait Huso, Amy Belton, David Huso, Linda M. S. Resar. High mobility group A1 chromatin remodeling protein expands the intestinal stem cell compartment and Paneth cell niche through Wnt/β-catenin signaling and Sox9. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1704.

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.

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.