Genetic Characterization of ABT-199 Sensitivity in Human AML

Abstract

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.

Venetoclax for AML: changing the treatment paradigm

“…Similar findings were reported by Bordeleau et al, who conducted a chemical screen using a collection of about 300 drugs on a cohort of 38 primary human AML specimens, and observed an association between mutations in IDH1/2 and sensitivity to venetoclax. 75 By producing R-2-hydroxyglutarate, IDH1/2 mutant cells inhibit the activity of cytochrome c oxidase in the mitochondrial electron transport chain, lowering the mitochondrial threshold to trigger apoptosis on engagement with venetoclax. Supporting this preclinical finding, 4 of 6 patients with relapsed refractory AML who responded to single-agent venetoclax in a phase 2 study carried an IDH1/2 mutation, and 12 of 16 patients with IDH1/2 mutation had a decrease in bone marrow blasts.…”

Sensitization effect of kaempferol from persimmon leaves on HepG2 hepatoma cells with ABT-199 resistance and its molecular mechanisms

“…But from Figure 6 we can see that compared with the negative control group, ABT-199 alone can significantly enhance the expression of Bax, and even significantly greater than in ABT-199 combined with KPL. This may be related to ABT-199’s ability to prevent the anti-apoptotic Bcl-2 from binding to pro-apoptotic Bax and Bak-1 proteins ( Bordeleau et al, 2018 ). This also shows that, although ABT-199 can up-regulate the expression of Bax protein, the inhibition of ABT-199 on Mcl-1, Bcl-xL and Bcl-w is very weak, so from our experimental results, we can see that ABT-199 alone has little effect on the apoptosis of HepG2 cells with high expression of Mcl-1.…”