Abstract 803: Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia

Abstract Background: Differentiation based therapy by all trans retinoic acid (ATRA) and arsenic trioxide (ATO) results in cure of >90% of patients with acute promyelocytic leukemia (APL). ATRA+ATO is highly biologically active in NPM1c AML, accounting for 30-40% of AML patients. ATO/ATRA induces proteasomal degradation of NPM1c, differentiation, growth arrest and TP53 dependent apoptosis in NPM1c cells. Furthermore, ATRA/ATO exposure restores nuclear localization of NPM1wt and significantly reduces blasts in NPMc AML patients. It was shown that the BET inhibitors OTX015/MK-8628 and JQ1 yield antileukemic activity and here we demonstrate their effects in NPM1c leukemia cells compared to ATRA/ATO. Methods : NPMc OCI-AML3 cell line or patient bone marrow (BM) blast cells obtained after informed consent were exposed to ATRA/ATO or OTX015/MK-8628 and JQ1. Apoptosis was assessed by annexin V/PI and caspase 3/PARP cleavage by WB. TP53 expression was detected by WB. Knock down of TP53 was performed with siRNA. Differentiation of OCI-AML3 cells was studied by CD11b surface expression and morphologic studies after MGG stain. Gene expression profiling was performed with GeneChip Array (Affymetrix®). NPMc expression was assessed by WB (+/- bortezomib) and cellular localization of NPMc/NPMwt was studied by immunofluorescence. Results : Exposure of OCI-AML3 cells to OTX015/MK-8628 and JQ1 was more potent to induce apoptosis as compared to ATRA/ATO. All treatments lead to caspase 3 and PARP cleavage. In OCI-AML3 cells, ATO-ATRA induced strong upregulation of genes of the TP53 dependent pathway (BAX/GADD45) while the anti apoptotic gene BCL2 was downregulated. In contrast, treatment with BET inhibitors lead to strong down regulation of the TP53 dependent pathway. In line, ATRA/ATO induced TP53 protein expression and TP53 knock down by siRNA decreased significantly ATRA/ATO induced apoptosis suggesting that apoptosis induced by BET inhibitors is TP53 independent. As compared to ATRA/ATO, OTX015/MK-8628 and JQ1 were more potent to induce differentiation as detected by CD11b surface expression and by morphologic analysis of OCI-AML3 cells. Interestingly, gene expression profiling of human leukocyte differentiation pathways in OCI-AML3 cells revealed different expression profiles for exposure to BET inhibitors compared to ATRA/ATO. Treatment of OCI-AML3 cells either by OTX015/MK8628, JQ1 or ATRA/ATO lead to proteosomal degradation of the NPMc protein. Exposure of OCI-AML3 cells and primary BM blasts of patients either by OTX015/MK8628, JQ1 or ATRA/ATO led to nuclear relocalization of NPMwt protein to the nucleus. Conclusion : BET inhibitors induce TP53 independent apoptosis, differentiation, proteasomal degradation and NPMwt relocalization in NPMc cells. Thus, clinical testing of bromodomain inhibitors in NPMc AML is indicated. Citation Format: Thorsten Braun, Marie-Magdelaine Coudé, Jeannig Berrou, Hanene Djamai, Mélanie Dupont, Anna Kaci, Marc Delord, Raphael Itzykson, Emmanuel Raffoux, Caroline Berthier, Hugues de Thé, André Baruchel, Claude Gardin, Hervé Dombret. Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 803

Biological Effects of BET Inhibition by OTX015 (MK-8628) and JQ1 in NPM1-Mutated (NPM1c) Acute Myeloid Leukemia (AML)

BET inhibitors (BETi) including OTX015 (MK-8628) and JQ1 demonstrated antileukemic activity including NPM1c AML cells. Nevertheless, the biological consequences of BETi in NPM1c AML were not fully investigated. Even if of better prognosis AML patients with NPM1c may relapse and treatment remains difficult. Differentiation-based therapy by all trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) demonstrated activity in NPM1c AML. We found that BETi, similar to ATO + ATRA, induced differentiation and apoptosis which was TP53 independent in the NPM1c cell line OCI-AML3 and primary cells. Furthermore, BETi induced proteasome-dependent degradation of NPM1c. BETi degraded NPM1c in the cytosol while BRD4 is degraded in the nucleus which suggests that restoration of the NPM1/BRD4 equilibrium in the nucleus of NPM1c cells is essential for the efficacy of BETi. While ATO + ATRA had significant biological activity in NPM1c IMS-M2 cell line, those cells were resistant to BETi. Gene profiling revealed that IMS-M2 cells probably resist to BETi by upregulation of LSC pathways independently of the downregulation of a core BET-responsive transcriptional program. ATO + ATRA downregulated a NPM1c specific HOX gene signature while anti-leukemic effects of BETi appear HOX gene independent. Our preclinical results encourage clinical testing of BETi in NPM1c AML patients

Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL

International audiencePrognosis of Philadelphia-positive (Ph(+)) acute lymphoblastic leukemia (ALL) in the elderly has improved during the imatinib era. We investigated dasatinib, another potent tyrosine kinase inhibitor, in combination with low-intensity chemotherapy. Patients older than age 55 years were included in the European Working Group on Adult ALL (EWALL) study number 01 for Ph(+) ALL (EWALL-PH-01 international study) and were treated with dasatinib 140 mg/day (100 mg/day over 70 years) with intrathecal chemotherapy, vincristine, and dexamethasone during induction. Patients in complete remission continued consolidation with dasatinib, sequentially with cytarabine, asparaginase, and methotrexate for 6 months. Maintenance therapy was dasatinib and vincristine/dexamethasone reinductions for 18 months followed by dasatinib until relapse or death. Seventy-one patients with a median age of 69 years were enrolled; 77% had a high comorbidity score. Complete remission rate was 96% and 65% of patients achieved a 3-log reduction in BCR-ABL1 transcript levels during consolidation. Only 7 patients underwent allogeneic hematopoietic stem cell transplantation. At 5 years, overall survival was 36% and up to 45% taking into account deaths unrelated to disease or treatment as competitors. Thirty-six patients relapsed, 24 were tested for mutation by Sanger sequencing, and 75% were T315I-positive. BCR-ABL1(T315I) was tested by allele-specific oligonucleotide reverse transcription-quantitative polymerase chain reaction in 43 patients and detection was associated with short-term relapses. Ten patients (23%) were positive before any therapy and 8 relapsed, all with this mutation. In conclusion, dasatinib combined with low-intensity chemotherapy was well-tolerated and gave long-term survival in 36% of elderly patients with Ph(+) ALL. Monitoring of BCR-ABL1(T315I) from diagnosis identified patients with at high risk of early relapse and may help to personalize therap