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Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction., SPI1 fusion genes in T-cell acute lymphoblastic leukemia (T-ALL) are commonly found with co-occurring NRAS mutations. Here, the authors show that the combination of these oncogenes is necessary to drive T-ALL in a murine model and that the oncogenic activity of the SPI1 fusion is dependent on β-catenin.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells. Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616–31. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517

FLT3 and PDGFR tyrosine kinases are important targets for therapy of different types of leukemia. Several FLT3/PDGFR inhibitors are currently under clinical investigation for combination with standard therapy for treatment of acute myeloid leukemia (AML), however these agents only induce partial remission and development of resistance has been reported. In this work we describe the identification of potent and novel dual FLT3/PDGFR inhibitors that resulted from our efforts to screen a library of 25,607 small molecules against the FLT3 dependent cell line MOLM-13 and the PDGFR dependent cell line EOL-1. This effort led to the identification of five compounds that were confirmed to be active on additional FLT3 dependent cell lines (cellular EC50 values between 35 and 700 nM), while having no significant effect on 24 other tyrosine kinases.

The FLT3 receptor tyrosine kinase is constitutively activated by internal tandem duplication (ITD) in its juxtamembrane domain or tyrosine kinase domain in 30% of acute myeloid leukemia (AML) cases.[1][1]-[3][2] Alternatively, FLT3 can also be activated by mutations in the kinase domain (such as the

Activation mutations in JAK3 occur in 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases, and co-occur frequently with HOXA cluster rearrangement. Genomic rearrangement of the HOXA cluster results in increased expression of HOXA9 and HOXA10. However it remains unclear if either HOXA9 or HOXA10 can cooperate with activating JAK3 mutations during oncogenic transformation and leukemogenesis. We have previously shown that JAK3 mutations lead to cell transformation and cause a long latency T-ALL in vivo using a mouse bone marrow transplant model. In this study we demonstrate that co-expression of the activating JAK3(M511I) protein with HOXA9 cooperate to develop leukemia within 30 days of transplant using an in vivo bone marrow transplant model. In our cooperative model, murine hematopoietic stem / progenitor cells were co-transduced with either both retroviral vectors encoding JAK3(M511I)/GFP and HOXA9/mCherry or each individually and then injected into sub-lethally irradiated recipient mice. Mice transplanted with bone marrow cells expressing JAK3(M511I) mutant alone developed T-ALL in 120 to 150 days. In sharp contrast, mice transplanted with cells expressing both JAK3(M511I) and HOXA9 showed rapid leukemia development within 30 days after transplant. Leukemia development was characterized by the rapid and specific increase in GFP-mCherry double positive cells. These animals showed high WBC, and splenomegaly and accumulation of immature CD8 single positive cells in the thymus. Similar experiments with HOXA10 did not show cooperation suggesting that HOXA9 is the more important oncogene in HOXA rearranged leukemias when a JAK3 activating mutation is present. To determine the underlying genetic mechanism for cooperation between HOXA9 and JAK3(M511I) the single positive JAK3 and double positive JAK3/HOXA9 expressing cells were isolated from thymi of leukemic mice for both epigenomic profiling using ATAC-seq and gene expression profiling. These analyses identified genetic pathways activated by the co-expression of HOXA9 and JAK3(M511I) mutation and provide mechanistic insight into the synergistic interaction between these two factors in driving leukemia development. Treatment of the animals with a JAK kinase inhibitor resulted in delayed leukemia development, confirming that the leukemia cells remain sensitive to the JAK inhibitor. This mouse model provides insight in the cooperation between oncogenes in leukemia development and provides a model for the study of targeted agents in this setting. Disclosures No relevant conflicts of interest to declare.