Dissecar o papel de HOXA9 em leucemia linfoblástica aguda de células B: para além dos cânones

Our solid preliminary data demonstrate that HOXA9 is downregulated in mouse B-ALL driven by IL7R mutational activation and in most human B-ALL subtypes, including those with enrichment for IL7R mutations, and that HOXA9 overexpression decreases the fitness of B-ALL cells (please see “Preliminary data” PDF file in section 13 of this application). Thus, we question the established notion that HOXA9 is strictly oncogenic during hematopoietic development and hypothesize that HOXA9 can also act as a tumor suppressor gene (TSG), protecting developing B-cells against the development of most B-ALL subtypes, with the notable exception of KMT2A-r and KMT2A-like ALL (where HOXA9 is often overexpressed and shown to promote leukemia development). To test and explore our hypothesis, we propose the following specific goals: AIM 1) to further validate that HOXA9 has a functionally-relevant role as TSG; AIM 2) to explore why and how HOXA9 acts as oncogene in KMT2A-r B-ALL and as TSG in other subtypes; AIM 3) to determine how HOXA9 is downregulated in B-ALL; AIM 4) to exploit therapeutic vulnerabilities in HOXA9-downregulated B-ALL. In AIM 1, we will use complementary strategies to confirm the tumor suppressor function of HOXA9 in the context of mouse mutant IL7R B-ALL development, namely by: a) evaluating the impact of overexpressing HOXA9 in additional leukemia samples; b) checking whether Hoxa9 downregulation at the pre-leukemic stage accelerates leukemia development; and c) determine HOXA9 deletion decreases leukemia latency and/or penetrance upon crossing of our CD2-Cre mutant IL7R mice with Hoxa-floxed mice [23]. In AIM 2, we will compare mouse mutant IL7R B-ALL versus mouse KMT2A-r B-ALL, and human ETV6::RUNX1 REH cells vs. human KMT2A-r RS4;11 cells. We will genetically modulate HOXA9 expression in these cells and evaluate impact on: differentiation, viability, proliferation, signaling, transcriptome, HOXA9 interacting partners, HOXA9 binding to the genome, and modulation of open chromatin regions. This will enable us to distinguish and contextualize the molecular features that underly oncogenic versus anti-tumoral functions of HOXA9. In AIM 3, we will explore whether epigenetic mechanisms such as DNA methylation or histone acetylation promote HOXA9 shutdown. Our preliminary data indicate that the histone deacetylase inhibitor SAHA/Vorinostat upregulates Hoxa9 in mouse mutant IL7R leukemias (Figure 2). We will also look at transcription factors (expression and binding to the HOXA9 locus) and histone marks known to regulate HOXA9 expression in: a) control, pre-leukemic and leukemic cells from mutant IL7R mice, and in b) KMT2A-r versus non-KMT2A-r cases (e.g. Ph-like and PAX5 P80R) human ALL patient samples. The region in chromosome 7 where HOXA9 is located was deleted in one B-ALL arising from mutant IL7R transduction of human hematopoietic progenitors [24], raising the possibility that this may occur in patients. Thus, we will also look for HOXA9 deletion or promoter/enhancer mutations. Finally, in AIM 4 we propose to explore the therapeutic potential of our findings by testing targetable alterations identified especially in AIM 2, both in vitro and in vivo. Our preliminary data showing that HOXA9 overexpression in mouse mutant IL7R B-ALLs leads to downregulation of PI3K/mTOR signaling (Figure 3) ensures that testing PI3K/mTOR inhibitors merits investigation.