Background: The HOXA9 protein is a transcription factor associated with normal hematopoiesis and which expression on hematopoietic stem cells and progenitor cells decreases in the course of blood cell differentiation. The expression of HOXA9 is however maintained at high level in several subtypes of acute myeloid leukemia (AML) such as mll ‐rearranged leukemias, NPM1‐mutated AMLs and AML presenting MYST3‐CREBP, NUP98‐NSD1 or RUNX/EVI1 translocations. The over‐expression of HOXA9 in these AML subtypes is associated with the maintenance of cell proliferation and the blockade of cell differentiation. It is well known that HOXA9 exerts its leukemic potential through DNA interaction and, therefore, we selected direct inhibitors of HOXA9/DNA interaction to block its activity and evaluate them on a murine leukemia cell model (Depauw et al., 2019). Aims: In order to go further, we aim at evaluating them on human AML cell models expressing or not HOXA9 and on normal human CD34+ hematopoietic cells to address any potential effect of our inhibitors on normal hematopoiesis. Methods: The expression level of HOXA9 was quantified by qRT‐PCR on a series of human AML cell lines. The sensitivity of those cell lines regarding two selected inhibitors was seen addressed using global cell survival analysis using MTS reaction. Methylcellulose clonogenic assays were performed on a series of human AML cell lines and on normal human bone marrow CD34+ hematopoietic cells. Knock‐down of HOXA9 expression was performed as a control using lentiviral delivery of HOXA9‐specific or control shRNAs. Results: A strong correlation between HOXA9 expression and the sensitivity to two of our inhibitors was evidenced in a series of 14 human AML cell lines with linear determination coefficients of R of ∼0.75 and ∼0.8. Clonogenic assays evidenced a decrease in the number and size of the colony forming units of treated related to untreated AML cell models. The relevance of HOXA9 expression in the clonogenicity is addressed by use of lentivirally transduced shHOXA9 in some of the AML cell models. By contrast, no difference were observed on either the number, the size and the differentiation subtypes of the colonies obtained from treated versus untreated normal human CD34+ hematopoietic cells. Summary/Conclusion: In conclusion, we selected inhibitors of HOXA9/DNA interaction that alter cell proliferation and clonogenic activity in a series of HOXA9‐positive AML cell models but that do not affect the progenitor cell number from normal bone marrow CD34+ cells.
Abstract CAR T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in AML remains elusive due to the risk of on-target off-tumor toxicity to hematopoietic stem and progenitor cells (HSPC) and insufficient T-cell persistence and longevity. Using a SynNotch circuit, we generated a high-precision “IF-THEN” gated logical circuit against the combination of CD33 and CD123 AML antigens and demonstrated anti-tumor efficacy against AML cell lines and patient-derived xenografts. Unlike constitutively expressed CD123 CAR-T cells, those expressed through the CD33 SynNotch circuit could preserve HSPCs and lower the risk of on-target off-tumor hematopoietic toxicity. These gated CAR-T cells exhibited lower expression of exhaustion markers (PD1, Tim3, LAG3, and CD39), higher frequency of memory T cells (CD62L+CD45RA+), and enhanced expansion. While targeting AML, the moderated circuit CAR signal also helped to mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy.
Le cancer du rein represente 3% des cancers de l’adulte. Il s’agit d’une pathologie tumorale heterogene insidieuse dont le pronostic reste generalement bon lorsque le cancer est decouvert precocement. Malheureusement, la survie a 5 ans ne depasse pas les 10% lorsque la decouverte se fait au stade metastatique. La prise en charge des patients repose sur la chirurgie et sur l’utilisation d’une therapie systemique aux stades avances ou metastatiques. Bien que ces dernieres annees, le cancer du rein ait fait l’objet d’une revolution therapeutique avec l’arrivee de therapies ciblees anti-angiogeniques et d’immunomodulateurs, les taux de reponse ne depassent pas les 30%. Il y a donc un interet tout particulier a identifier de nouvelles cibles therapeutiques dans ce cancer pour esperer de meilleurs traitements.Pour cela, nous travaillons sur le role du facteur de transcription HOXA9 et sur son inhibition par de petites molecules, capables d’entrer en competition avec sa liaison a l’ADN, d’inhiber son activite transcriptionnelle et, par consequent, les programmes oncogeniques qu’il regule. Deux molecules inhibitrices ont ete selectionnees.Dans un premier temps, nous avons utilise une base de donnees publique et montrequ’HOXA9 etait surexprime dans le sous-type papillaire et que son expression etait correlee a la survie des patients dans le sous-type a cellules claires.Puis, a l’aide de deux lignees cellulaires humaines representant les deux principaux types histologiques de carcinome a cellules renales, nous avons montre qu’elles exprimaientfortement HOXA9 et evalue ses effets biologiques par invalidation par ARN interference.Les effets biologiques observes ont ete compares a ceux obtenus par le traitement avecles molecules inhibitrices. Nous avons ainsi pu montrer que l’invalidation d’HOXA9entrainait la mort des cellules et impactait la migration cellulaire. De plus, le traitementpar les molecules inhibitrices impacte egalement la survie des cellules et leur migration.Pour identifier les genes regules indirectement ou directement par HOXA9, une analyse transcriptomique apres invalidation a identifie qu’HOXA9 regule des genes impliques dans la voie du TNFα, et notamment certains genes deja decrits dans le carcinome renal tels que BMP2. BMP2 etant connu pour reguler la sous-population de cellules souches cancereuses (CSC), nous avons evalue le role d’HOXA9 dans cette population et montrequ’HOXA9 est surexprime lors de la formation de tumorospheres, une caracteristique des CSC et que son invalidation abolit totalement la formation de ces spheres. Pour confirmer ces resultats, des cellules invalidees pour HOXA9 ont ete implantees orthotopiquement a des souris immunodeficientes et montrent qu’HOXA9 est necessaire a la tumorigenicite.Enfin, pour evaluer le role d’HOXA9 dans les metastases du cancer renal et leur ciblage potentiel, nous avons genere un modele metastatique pulmonaire et montre que l’invalidation d’HOXA9 est associee a une diminution de la formation de metastases pulmonaires du carcinome renal.
<div>Abstract<p>Chimeric antigen receptor (CAR) T-cell therapy has remarkably succeeded in treating lymphoblastic leukemia. However, its success in acute myeloid leukemia (AML) remains elusive because of the risk of on-target off-tumor toxicity to hematopoietic stem/progenitor cells (HSPC) and insufficient T-cell persistence and longevity. Using a SynNotch circuit, we generated a high-precision “IF-THEN” gated logical circuit against the combination of CD33 and CD123 AML antigens and demonstrated antitumor efficacy against AML cell lines and patient-derived xenografts. Unlike constitutively expressed CD123 CAR-T cells, those expressed through the CD33 SynNotch circuit could preserve HSPCs and lower the risk of on-target off-tumor hematopoietic toxicity. These gated CAR-T cells exhibited lower expression of exhaustion markers (PD-1, TIM-3, LAG-3, and CD39), higher frequency of memory T cells (CD62L<sup>+</sup>CD45RA<sup>+</sup>), and enhanced expansion. Although targeting AML, the moderated circuit CAR signal also helped mitigate cytokine release syndrome, potentially addressing one of the ongoing challenges in CAR-T immunotherapy.</p>Significance:<p>Our study demonstrates the use of “IF-THEN” SynNotch-gated CAR-T cells targeting CD33 and CD123 in AML reduces off-tumor toxicity. This strategy enhances T-cell phenotype, improves expansion, preserves HSPCs, and mitigates cytokine release syndrome—addressing critical limitations of existing AML CAR-T therapies.</p></div>
