The Hoxa9 and Meis1 genes represent important oncogenic collaborators activated in a significant proportion of human leukemias with genetic alterations in the MLL gene. In this study, we show that the transforming property of Meis1 is modulated by 3 conserved domains, namely the Pbx interaction motif (PIM), the homeodomain, and the C-terminal region recently described to possess transactivating properties. -HOXA9) that characterize myeloid leukemias in human and mice; (3) the overexpression of HOXA7 and HOXA9 in several MLL-induced human leukemias 11,12 ; (4) the distinctly high correlation between high levels of HOXA9 expression and poor prognosis in human acute myeloid leukemia (AML) 13 ; and (5) coexpression of BCR-ABL and NUP98-HOXA9 in a significant number of human leukemic specimens, 14 and a recent proof of their genetic interaction. 15 Together, these studies establish a direct and indirect role for multiple Hox genes in human leukemias, and highlight the importance of revealing the molecular bases of Hox-induced transformation.Some insight into the molecular mechanisms of Hox-induced transformation may be gained from studies of Hox cofactors, members of the TALE (for 3-amino-acid loop extension) family of homeodomain proteins. Of interest, founders of 2 subgroups within this family, namely PBX1 16,17 and Meis1, 18 were identified based on their participation in human and mouse leukemia, respectively.Hox, Pbx, and Meis participate in a multiprotein interaction network. The cooperative interaction between Pbx and Hox proteins enhances the DNA binding affinity and specificity of Hox proteins 19 and is essential for at least some of the Hox-dependent developmental programs. [20][21][22] In contrast, a functional role for a dimeric Hox-Meis complex has so far not been established. 23 Members of the Meis family can form complexes with Pbx in DNA-dependent and independent manners. [24][25][26] Interaction with Meis induces nuclear localization of Pbx by preventing its nuclear export 27,28 and promoting nuclear import. 29,30 Indirect interaction between Hox and Meis proteins was established by the identification of Hox-Pbx-Meis heterotrimeric complexes. 26 PBX1 is involved as part of the fusion E2a-PBX1 oncoprotein in a high proportion of human pre-B leukemias. 16,17 We recently generated a mouse model of E2a-PBX1-induced pre-B-cell leukemia, and showed that the Hoxa locus was targeted by murine Moloney leukemia virus (MMLV) in the majority of the leukemias analyzed. 31 These leukemias were characterized by aberrant expression of Hoxa genes, and all expressed high levels of Hoxa7, thus pointing to a genetic interaction between E2a-PBX1 and Hoxa genes. In support of these findings, we

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Graphical AbstractHighlights d ITGA3 + cells positively identify LT-repopulating HSCs in cultured cord blood cells d ITGA3 + cells display durable multipotentiality d ITGA3 + is functionally required for the LT-engraftment ability of CB cells SUMMARY Transplantation of expanded hematopoietic stem cells (HSCs) and gene therapy based on HSC engineering have emerged as promising approaches for the treatment of hematological diseases. Nevertheless, the immunophenotype of cultured HSCs remains poorly defined. Here, we identify Integrin-a3 (ITGA3) as a marker of cultured human HSCs. Exploiting the pyrimidoindole derivative UM171 to expand cord blood (CB) cells, we show that ITGA3 expression is sufficient to separate the primitive EPCR + CD90 + CD133 + CD34 + CD45RA À HSC population into two functionally distinct fractions presenting mostly short-term (ITGA3 À ) and both short-term and long-term (ITGA3 + ) repopulating potential. ITGA3 + cells exhibit robust multilineage differentiation potential, serial reconstitution ability in immunocompromised mice, and an HSC-specific transcriptomic signature. Moreover, ITGA3 expression is functionally required for the long-term engraftment of CB cells. Altogether, our results indicate that ITGA3 is a reliable marker of cultured human long-term repopulating HSCs (LT-HSCs) and represents an important tool to improve the accuracy of prospective HSC identification in culture.

The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX-and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1- MC IntroductionThere is a compelling body of evidence implicating the HOX transcription factors and their cofactors PBX and MEIS/PKNOX-(PREP) in leukemogenesis (reviewed by Argiropoulos et al 1 ). In particular, deregulated expression of HOXA9 has been detected in a large proportion of human acute myeloid (AML) and lymphoid leukemias (ALLs) [2][3][4][5] and is associated with poor prognosis for refractory AML. 2 A significant proportion of these leukemias, especially those harboring MLL rearrangements, also overexpress MEIS1, [3][4][5][6] indicating that activation of MEIS1 may represent a key collaborating genetic event in leukemia development.MEIS1 is a member of the TALE class of homeoproteins, which includes the PBX and MEIS/PKNOX protein families characterized by an atypical homeodomain (HD) containing a three-amino acid loop extension (TALE) between the first 2 ␣-helices. 7 MEIS1/PKNOX1 and PBX1 proteins physically interact with each other using the bipartite Homothorax-MEIS domain (HM A /HM B ) and the PBC-A domain of PBX. 8,9 This interaction was reported to enhance the stability of the heterodimers and to promote their nuclear localization. [10][11][12] MEIS1 and PBX1 also form heterodimers with HOX proteins in a DNAdependent manner and have been reported to form triple complexes composed of MEIS1/PKNOX1, PBX1, and HOX in both a DNA-independent 13,14 and DNA-dependent manner, 15 suggesting that, in some cellular contexts, a triple HOX-PBX-MEIS/PKNOX complex is required for HOX-mediated gene regulation. 15,16 The combinatorial diversity generated by 39 different Hox, 3 Meis, 2 Pknox, and 4 Pbx gene products probably enables formation of distinct regulatory complexes targeting numerous genomic loci in a cell context-dependent manner.Using a bone marrow transduction/transplantation-based experimental model, we showed that overexpression of Meis1 significantly accelerates the onset of Hoxa9-induced AML, 17,18 and similar collaborative Meis1 effects were reported for other Hox and Hox-fusion genes tested. 19,20 High levels of Meis1 expression also appeared to be the rate-limiting factor for development of Mllinduced leukemias, 21 implying that Meis1 activity is necessary to create and/or maintain cellular context required for tumor development. Pknox1 overexpression, in contrast, fai...

Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.

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Acute myeloid leukemias (AML) are aggressive blood cancers characterized by an overall survival of 27% at 5 years. The main challenge in AML treatment originates from the genetic heterogeneity of the disease that contributes to the wide range of clinical outcomes observed. A large proportion (~35%) of AML patients exhibit no distinguishable chromosomal abnormalities that can be used to guide treatment selection and are therefore classified in the poorly characterized intermediate risk category. Approximately 50-60% of intermediate risk AML patients carry mutations in the NPM1 gene. These mutations are associated with a favorable outcome unless a concomitant mutation in the FLT3 gene is detected, which accounts for 39% of cases. The survival rate is further worsened when a third mutation is detected in the DNMT3A gene, dropping from 40% to 20% 5 years post-treatment for double and triple mutants, respectively. This study aimed to identify drugs selectively affecting the viability of leukemic cells from AML patients with NPM1 mutations. To achieve this, we took advantage of culture conditions developed by our group that prevent differentiation of leukemic cells and preserve leukemia stem cell activity from primary AML specimens (Pabst et al., Nature methods, 2014), enabling chemical screening of primary AML specimens. We conducted a chemical screen using a collection of ~300 clinical grade drugs on a cohort of 38 primary human AML specimens containing NPM1 mutated (NPM1c+) and NPM1 wild-type (NPM1wt) samples. These specimens belonged to the Leucegene collection of sequenced and clinically annotated samples. The screen identified ABT-199 as the compound with the most discriminatory effect toward NPM1c+ AML. ABT-199 is a specific BH3-mimetic that prevents anti-apoptotic BCL2 from binding pro-apoptotic BAX and BAK1 proteins, leading to apoptosis. ABT-199 demonstrated encouraging results for AML treatment, but the determinants of drug sensitivity have not been well defined. Analysis of the enrichment of clinical variables in ABT-199 sensitive and resistant groups of AML specimens to define characteristics/biomarkers associated with ABT-199 sensitivity in AML revealed that mutations in NPM1, RAD21, IDH1, IDH2, DNMT3A and FLT3 (ITD), as well as normal karyotype and the FAB M1 class all significantly associate with increased ABT-199 sensitivity. At the other side of the spectrum, mutations in TP53 and FAB class M5B were significantly enriched in the resistant group. Additional analyses revealed that NPM1c+/DNMT3Amut/FLT3-ITD specimens are sensitive to ABT-199, which may provide a rationale to prioritize patients from this adverse risk AML subgroup for explorative ABT-199 based regimens. Specimens with RAD21 mutations were the most sensitive to ABT-199 treatment and further analyses demonstrated a clear association between mutation of cohesin genes (RAD21, SMC1A, SMC3, STAG2) and increased ABT-199 sensitivity. In line with this, we demonstrated that RAD21 knockdown alone is able to sensitise AML cell lines to BCL2 inhibition. Comparative transcriptome analysis of ABT-199 sensitive and resistant specimens also revealed an apoptotic gene signature linked to ABT-199 resistance with BCL2A1, an anti-apoptotic BCL2 homolog, being the most differentially expressed apoptotic gene between these response groups and showing increased expression in the resistant subset. Expression correlation analysis over the 415 specimens of the Leucegene cohort showed that BCL2A1 is one of the top genes anti-correlated to BCL2, and accordingly, high BCL2 and BCL2A1 expressors were enriched among ABT-199 sensitive and resistant specimens, respectively. In conclusion, using an unbiased pharmacogenomic approach, we identified ABT-199, a compound with the potential to eradicate NPM1c+ AML, which has already been tested in a phase 2 clinical trial for AML. Our results shed light on determinants of ABT-199 sensitivity which could readily impact AML therapy by providing a rationale for prioritizing patients with NPM1, RAD21, IDH1 and/or IDH2 mutations for ABT-199 AML trials. Our results also uncover potential mechanisms of resistance to ABT-199, providing grounds to design combination therapies to overcome ABT-199 chemoresistance. Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

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