Final Report Summary - TIGRE (Targeting glucocorticoid resistance in T-ALL: a Systems Biology approach)
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer that accounts for 15% of pediatric and 25% of adult ALL cases. Despite recent progress of clinical outcomes in this disease, 25% of children and over 50% of adult T-ALL cases show primary resistant disease or respond only transiently to chemotherapy and relapse. Thus, a major understanding of the mechanism of drug resistance and of the molecular basis of T-ALL pathogenesis is necessary to further advance in the treatment of T-ALL. Over the past few years, different research groups identified novel mutations of key genes involved in the pathogenesis of T-ALL and demonstrated that inactivation of NOTCH1 signaling can reverse glucocorticoid resistance in specific T-ALLs. Despite numerous studies addressing the mechanisms that mediate the response of lymphoid tumor cells to glucocorticoid therapy, the molecular basis of glucocorticoid resistance remain incompletely understood as glucocorticoid resistance is probably mediated by the combination of multiple primary oncogenic lesions responsible for T-cell transformation and secondary mutations selected during glucocorticoid therapy. Based on the observation that NOTCH1 signaling promotes cell growth proliferation and survival in immature T-cells, a phenotype opposed to that of glucocorticoids, I hypothesized that aberrant NOTCH1 signaling might contribute to glucocorticoid resistance in T-ALL. Following on this hypothesis, the principal aim of this work is to identify novel genes and pathways involved in T-cell transformation and glucocorticoid resistance in T-ALL using cutting edge experimental methods and reverse engineering of transcriptional networks.
During the outgoing phase of this fellowship, I first studied the oncogenic programs controlled by TLX1 and TLX3, two transcription factors activated by oncogenic chromosomal translocations in 30% of adult and pediatric T-ALLs, I cross-analyzed the gene expression signatures of human tumors with activation of TLX1 and TLX3 with those of TLX1-induced mouse T-ALLs. Integration of these analyses with cytogenetic and cellular assays demonstrated that malignant activation of TLX1 and TLX3 disrupt the mitotic checkpoint and induce aneuploidy at the earliest stages of leukemic transformation. To further characterize the genetic circuitries controlled by TLX1 and TLX3 in T-ALL I examined the genetic regulatory programs controlled by these transcription factor oncogenes using a combination of ChIP-chip analyses, gene expression profiling and reverse engineering of transcriptional networks derived from a comprehensive microarray series of 228 primary T-ALL samples. These analyses identified RUNX1 as major regulator of the oncogenic program controlled by TLX1 and TLX3 and supported a tumor suppressor role for this transcription factor in the pathogenesis of T-ALL. This hypothesis was fully validated by mutation analysis of RUNX1 in a broad panel of T-ALL primary samples, which revealed the presence of recurrent loss of function mutations in RUNX1 in T-ALL. Interestingly, RUNX1 mutants are associated to a newly characterized ALL subtype known as “early T cell precursor” ETP-ALL characterized by high risk of treatment failure and poor prognosis. To ascertain whether Runx1 inactivation can serve as an initiating molecular lesion in the pathogenesis of T-ALL, I recently performed ENU mutagenesis of Runx1 wild type and Runx1+/- haploinsufficient mice. Strikingly, this experiment demonstrated a marked shift in the immunophenotype of tumors developed after ENU mutagenesis from a typical CD4/CD8 double-positive cortical T-ALL in Runx1+/+ mice to ETP-TALL in Runx1+/- animals. Interestingly, Runx1 mutations and expression analyses demonstrated retention of wild type Runx1 in these tumors and haploinsufficient expression of this tumor suppressor gene. Altogether, these analyses identified RUNX1 as a critical haploinsufficient tumor suppressor gene in ETP T-ALL. Finally, during the return phase of this fellowship, I focused my attention on the bioinformatics analysis of the oncogenic pathways controlled by NOTCH1 driving T-cell transformation and leading to glucocorticoid treatment resistance. Indeed, during the outgoing phase I also mapped and characterized the genomic landscape of NOTCH1 direct target genes in T-ALL using ChIP-seq analysis. I thus developed a bioinformatics pipeline to integrate these Chip-seq with gene expression profiling analyses of T-ALL tumors treated with gamma secretase inhibitors and normal thymocyte populations at different stages of human T-cell development. I was thus able to identify a wide range of new NOTCH1 functional targets involved in T-cell transformation. In addition, these studies supported a critical role for ETS1 and RUNX1 as major modulators and co-regulators of NOTCH1 target genes. Detailed ChIP-seq and expression analyses of RUNX1 and ETS1 in T-ALL and analysis of RUNX1, ETS1 and NOTCH1 knockout and conditional knockout mice have formally demonstrated a strict requirement of these transcription factors in the regulation of NOTCH1 target genes and in the generation and maintenance of NOTCH1 induced leukemia. Finally these studies suggest that development and use of RUNX1 and ETS1 inhibitors could be a valid therapeutic alternative in the treatment of mature T-ALL.
I believe that the advancement toward the objectives of this research is in full agreement with my project proposal. This fellowship gave me the opportunity to greatly improve my scientific maturity and enhance my interdisciplinary scientific background. Moreover, thanks to a comprehensive training in research manuscripts preparation and grant writing, I greatly improved my scientific writing skills and the ability to elaborate hypothesis driven research proposals. All this helped in building a definitive commitment to the strongest scientific integrity, which makes me ready to start my career as independent researcher at my home institution. Toward this goal in my future plans is to apply for a Telethon Career Award Program at the Dulbecco Telethon Institute and other Italian and European funding agencies (i.e. Italian Ministry of Education, Universities and Research –MIUR or European Research Council- ERC) in order to obtain an adequate funding for my new independent research group.
During the outgoing phase of this fellowship, I first studied the oncogenic programs controlled by TLX1 and TLX3, two transcription factors activated by oncogenic chromosomal translocations in 30% of adult and pediatric T-ALLs, I cross-analyzed the gene expression signatures of human tumors with activation of TLX1 and TLX3 with those of TLX1-induced mouse T-ALLs. Integration of these analyses with cytogenetic and cellular assays demonstrated that malignant activation of TLX1 and TLX3 disrupt the mitotic checkpoint and induce aneuploidy at the earliest stages of leukemic transformation. To further characterize the genetic circuitries controlled by TLX1 and TLX3 in T-ALL I examined the genetic regulatory programs controlled by these transcription factor oncogenes using a combination of ChIP-chip analyses, gene expression profiling and reverse engineering of transcriptional networks derived from a comprehensive microarray series of 228 primary T-ALL samples. These analyses identified RUNX1 as major regulator of the oncogenic program controlled by TLX1 and TLX3 and supported a tumor suppressor role for this transcription factor in the pathogenesis of T-ALL. This hypothesis was fully validated by mutation analysis of RUNX1 in a broad panel of T-ALL primary samples, which revealed the presence of recurrent loss of function mutations in RUNX1 in T-ALL. Interestingly, RUNX1 mutants are associated to a newly characterized ALL subtype known as “early T cell precursor” ETP-ALL characterized by high risk of treatment failure and poor prognosis. To ascertain whether Runx1 inactivation can serve as an initiating molecular lesion in the pathogenesis of T-ALL, I recently performed ENU mutagenesis of Runx1 wild type and Runx1+/- haploinsufficient mice. Strikingly, this experiment demonstrated a marked shift in the immunophenotype of tumors developed after ENU mutagenesis from a typical CD4/CD8 double-positive cortical T-ALL in Runx1+/+ mice to ETP-TALL in Runx1+/- animals. Interestingly, Runx1 mutations and expression analyses demonstrated retention of wild type Runx1 in these tumors and haploinsufficient expression of this tumor suppressor gene. Altogether, these analyses identified RUNX1 as a critical haploinsufficient tumor suppressor gene in ETP T-ALL. Finally, during the return phase of this fellowship, I focused my attention on the bioinformatics analysis of the oncogenic pathways controlled by NOTCH1 driving T-cell transformation and leading to glucocorticoid treatment resistance. Indeed, during the outgoing phase I also mapped and characterized the genomic landscape of NOTCH1 direct target genes in T-ALL using ChIP-seq analysis. I thus developed a bioinformatics pipeline to integrate these Chip-seq with gene expression profiling analyses of T-ALL tumors treated with gamma secretase inhibitors and normal thymocyte populations at different stages of human T-cell development. I was thus able to identify a wide range of new NOTCH1 functional targets involved in T-cell transformation. In addition, these studies supported a critical role for ETS1 and RUNX1 as major modulators and co-regulators of NOTCH1 target genes. Detailed ChIP-seq and expression analyses of RUNX1 and ETS1 in T-ALL and analysis of RUNX1, ETS1 and NOTCH1 knockout and conditional knockout mice have formally demonstrated a strict requirement of these transcription factors in the regulation of NOTCH1 target genes and in the generation and maintenance of NOTCH1 induced leukemia. Finally these studies suggest that development and use of RUNX1 and ETS1 inhibitors could be a valid therapeutic alternative in the treatment of mature T-ALL.
I believe that the advancement toward the objectives of this research is in full agreement with my project proposal. This fellowship gave me the opportunity to greatly improve my scientific maturity and enhance my interdisciplinary scientific background. Moreover, thanks to a comprehensive training in research manuscripts preparation and grant writing, I greatly improved my scientific writing skills and the ability to elaborate hypothesis driven research proposals. All this helped in building a definitive commitment to the strongest scientific integrity, which makes me ready to start my career as independent researcher at my home institution. Toward this goal in my future plans is to apply for a Telethon Career Award Program at the Dulbecco Telethon Institute and other Italian and European funding agencies (i.e. Italian Ministry of Education, Universities and Research –MIUR or European Research Council- ERC) in order to obtain an adequate funding for my new independent research group.