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Abstract 3969 Poster Board III-905 Because T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid leukemia with a high rate of relapse in both children and adults, research is urgently needed to discover molecular pathways that could be targeted in therapy. Recent studies have identified NOTCH1 activating mutations, genomic classifiers, and other molecular pathways that are disrupted in T-ALL, although these findings have not yet translated into better therapy or improved long-term survival. Thus, we are investigating molecular pathways that play a causative role in T-ALL and could be modulated by therapy. We previously showed that HMGA1a is overexpressed in human T-ALL. Further, HMGA1a transgenic mice develop aggressive T-ALL with complete penetrance, indicating that HMGA1a induces leukemogenic transformation in vivo. Disruption of the CDKN2A (INK4A/ARF) tumor suppressor locus has been reported in up to 90% of T-ALL, suggesting that this contributes to leukemic transformation in T-cells. To determine if loss of function of the INK4A/ARF tumor suppressor locus could cooperate with HMGA1a in T-ALL, we crossed our HMGA1a transgenics with mice that are null for the INK4A/ARF locus. We found that T-ALL was markedly accelerated in the HMGA1a transgenics that are also null at this tumor suppressor locus. These mice died significantly earlier than the HMGA1 transgenics that are wildtype at the INK4A/ARF locus (4.8 months+/- 1.0 month versus 10.5 +/- 2.5 months, p < 0.0001 by the student's t-test). The HMGA1a transgenic, INK4A/ARF null mice recapitulate salient clinical and pathologic features of human T-ALL. All mice appeared ill and exhibit marked organomegaly with extensive leukemic infiltration. Immunophenotyping by fluorescence activated cell sorting showed T-ALL in all cases with identical T-cell markers (CD3, CD8, and αβ-TCR) to those previously reported for the HMGA1a transgenics at 8-10 months. Interestingly, the HMGA1a-INK4A/ARF+/- mice also developed early lymphoid malignancy in 6/17 cases. The accelerated lymphoid malignancy in these mice suggests that there is loss of heterozygosity at the INK4A/ARF locus. Alternatively, haploinsufficiency of INK4A and/or ARF may be sufficient to promote lymphoid tumorigenesis in the setting of HMGA1a overexpression. To determine if HMGA1 overexpression and loss of INK4A/ARF expression occur in human T-ALL, we analyzed gene expression profile analysis in leukemic blasts from children and young adults with T-ALL. We found high levels of HMGA1 expression and low levels of INK4A/ARF expression in most patients. Taken together, our results indicate that overexpression of HMGA1 and loss of function at the INK4A/ARF (CDKN2A) tumor suppressor locus cooperate in T-cell leukemogenesis and these pathways could represent rational therapeutic targets. Further, our mice provide a novel preclinical model for T-ALL. Disclosures: No relevant conflicts of interest to declare.

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.

While the oncogenic properties of the high mobility group A1 (HMGA1) gene are well established, the molecular pathways that mediate transformation by HMGA1 have not been clearly defined. HMGA1 is widely overexpressed in hematologic malignancies and other human cancers. Moreover, its overexpression portends a poor prognosis in some tumors. The HMGA1 gene encodes the HMGA1a and -A1b chromatin binding proteins. We previously showed that overexpression of HMGA1a or -A1b confers a transformed phenotype in cultured, human lymphoid cells. More recently, we developed transgenic mice overexpressing HMGA1a and all mice develop aggressive lymphoid malignancy. Because HMGA1 proteins function in regulating gene expression, we hypothesize that overexpression of HMGA1 leads to neoplastic transformation by altering expression of specific target genes involved in this process. To identify genes that are regulated by HMGA1 in transformation, we used microarray gene expression profile analysis in cells transfected to overexpress HMGA1a or -A1b. Ninety-seven genes were differentially regulated by ≥ 2-fold in cells overexpressing HMGA1a and 103 genes were differentially regulated by ≥ 2-fold in cells overexpressing HMGA1b. Fifty-five of these genes were common to both the HMGA1a and -A1b cells. From the list of genes up-regulated in cells overexpressing HMGA1a or A1b, the gene encoding the signaling molecule, signal transducer and activator of transcription or STAT3, was studied further because of its prominent role in malignancy. HMGA1 binds to the STAT3 promoter in human leukemia cells in vivo by chromatin immunoprecipitation experiments and enhances transcription of the STAT3 promoter in transfection experiments. Both total STAT3 and activated STAT3 proteins are increased in leukemia cells from HMGA1a transgenic mice compared to lymphoid cells from control mice. Moreover, activated STAT3 recapitulates the transforming activity of HMGA1a in cultured cells. Blocking STAT3 function preferentially induces apoptosis in leukemia cells from HMGA1a transgenic mice, but not in control cells. STAT3 mRNA is also increased in human acute lymphoid leukemia samples that overexpress HMGA1a, suggesting that this pathway is also involved in human lymphoid leukemia. Our results indicate that STAT3 is a direct HMGA1a target gene that is both necessary and sufficient for transformation induced by HMGA1a.