Venetoclax and arsenic showed synergistic anti‐leukemia activity in vitro and in vivo for acute myeloid leukemia with the NPM1 mutation

For patients with relapsed and/or refractory (R/R) acute myeloid leukemia (AML), available therapies are limited, and cure rates remain less than 10%.1 Novel drugs targeting this population are eagerly awaited. With B-cell lymphoma 2 (BCL-2) protein overexpression is implicated in the survival of AML cells and chemotherapy resistance.2, 3 Therefore, BCL-2 inhibitors, such as venetoclax, are good candidates for inducing apoptosis and overcoming resistance. Moreover, arsenic-mediated induction of reactive oxygen species induces both caspase-dependent and caspase-independent apoptosis.4, 5 The combination of BCL-2 inhibitors and arsenic has been proposed to synergistically induce apoptosis in AML cells, although this theory has never been demonstrated in vitro or in vivo. Herein, we demonstrated, for the first time, that arsenic and venetoclax could synergistically induce the apoptosis of NPM1-mutated OCI AML-3 cells in vitro. Moreover, we definitively demonstrated the anti-leukemia activity of venetoclax and oral arsenic in two R/R AML patients with NPM1-mutations. The OCI-AML3 cell line was used to evaluate whether BCL-2 inhibitors and arsenic synergistically induce apoptosis in AML cells with the NPM1 mutation. Cell proliferation was determined by the MTT assay. As shown in Figure 1A, OCI-AML3 cells exhibited high sensitivity to ATO and moderate sensitivity to venetoclax. However, the combination of ATO and venetoclax significantly inhibited cell proliferation compared with ATO or venetoclax monotherapies. Furthermore, changes in apoptosis-associated protein expression levels were detected in OCI-AML3 cells following treatment with venetoclax with or without ATO. When cells were treated with venetoclax monotherapy, cleaved-PRAP and cleaved-caspase 3 increased, and phosphorylated AKT decreased in a dose-dependent manner. However, no appreciable changes were observed in NPM1 protein expression (Figure 1B). Comparatively, the combination therapy with ATO significantly enhanced the generation of cleaved-PRAP and cleaved-caspase 3 (Figure 1C), which suggests venetoclax and ATO synergistically promote the apoptosis of OCI-AML3 cells. To further confirm the above results, the apoptosis of OCI-AML3 cells following venetoclax and/or ATO treatment was further evaluated by flow cytometry. Consistent with the MTT and western blot results, the combination venetoclax and ATO treatment group showed remarkably higher rates of apoptosis than the single treatment groups (Figure 1D). Additionally, we also determined the morphological changes in the NPM1 and PML proteins after ATO and/or venetoclax treatment. Confocal microscopy revealed that the PML protein is located in the nucleus of OCI-AML3 cells as smaller and heterogenous nuclear bodies (NBs). The ATO monotherapy or combination therapy with venetoclax markedly facilitated the restoration of macrospeckled PML NBs and PML protein degradation. The NPM1 protein is mainly distributed in the nucleus, and no appreciable changes were observed after treatment. Collectively, the results obtained from the in vitro experiments revealed that ATO and venetoclax indeed exert synergistic effects on OCI-AML3 cells carrying the NPM1 mutation. Therefore, we subsequently validated the synergistic anti-leukemia activity in vivo. Two R/R NPM1-mutated AML patients were recruited in our study and assigned to treatment with venetoclax and oral arsenic. Case 1: A 60-year-old male was admitted on 23 March 2019, because he relapsed after 8 years of AML with the NPM1 and IDH2 mutations. He had a white blood cell count of 6.6 × 109/L; a hemoglobin level of 114 g/L; and a platelet count of 189 × 109/L. A bone marrow (BM) smear showed hypercellularity with 82% blasts. The leukemia cells expressed CD117, CD33 and CD13 but did not express CD34 or other T- or B-lymphoid-related markers. The patient harbored the NPM1 and IDH2 mutations. Karyotype analysis revealed 46, XY [20]. Because an acute cerebral infarction occurred, he received venetoclax (100 mg d1, 200 mg day 2 to day 28) and oral arsenic in the form of the Realgar-Indigo naturalis formula (RIF) (60 mg/kg daily in an oral divided dose on day2~15). Informed consent was obtained from the patient. CR was achieved after 28 days, and FCM showed MRD at 0.041%. He received consolidation therapy, which included two cycles of venetoclax plus azacitidine and two cycles of idarubicin plus cytarabine. He maintained CR for 6 months until October 2019. Case 2: A 29-year-old female was admitted on November 26, 2018, due to R/R AML with the NPM1 and IDH1 mutations. She had a white blood cell count of 2.0 × 109/L; a hemoglobin level of 66 g/L; and a platelet count of 27 × 109/L. A bone marrow smear showed hypocellularity with 34% blasts, and the NPM1-m transcript level was 4.8%. She was first diagnosed 22 months prior to admission and achieved CR with an incomplete count recovery (CRi) after induction therapy with the CAG regimen (AraC, aclamycin and G-CSF). Because of severe infection, a history of long-term thrombocytopenia and the inability to receive standard chemotherapy, she suffered two relapses during the first year. She agreed to receive venetoclax (100 mg on d1 and 200 mg on d2~28) because of concurrent voriconazole use to control a lung infection. Informed consent was obtained from the patient. At 28 days (December 28, 2018), the bone marrow blasts decreased to 2.5%; minimal residual disease (MRD) by flow cytometry was negative (acquired 750 000 cells); and NPM1-m transcript levels decreased to 0.011%. Complete remission with an incomplete count recovery (CRi) was achieved. To enhance apoptosis, RIF (60 mg/kg daily in an oral divided dose) was combined with venetoclax for outpatient treatment during the second and third months, and the patient maintained CRi status for 3 months. Flow cytometry was negative for MRD, and the NPM1-m transcript level was 0.01% and 0.04% in the second and third months, respectively. She relapsed at 4 months after achieving a CR, received a haploidentical hematopoietic stem cell transplantation as salvage treatment, and was still alive and in CR as of October 2019. To date, the patient has been alive for 30 months since first diagnosis. The two cases contained NPM1 and IDH1/2 mutations and showed high sensitivity to venetoclax monotherapy or venetoclax and oral arsenic combination therapy. This is consistent with a recent study showing a CR + CRi rate of 91% and 71% for NPM1 and IDH1/2 mutated subtypes, respectively, treated with venetoclax and a hypomethylating agent in newly diagnosed elderly AML patients.6, 7 Previous studies demonstrated that ATO and ATRA target the NPM1 mutant oncoprotein and induce apoptosis in NPM1-mutated AML cells.8, 9 We demonstrated that arsenic and venetoclax could synergistically induce AML cell apoptosis in vitro, which needs to be further validated. According to the MRD results of our two patients, it seems that only one cycle of arsenic and venetoclax combination therapy was capable of inducing a similar response to that observed in the second case, which added oral arsenic after the first cycle of venetoclax monotherapy. In theory, the combined use of arsenic and venetoclax may be better considering the synergistic induction of apoptosis by the two drugs. Our study provided preliminary evidence to support the combination of venetoclax and arsenic in the treatment of NPM1-mutated AML. Because oral arsenic is commercially available in China, a completely oral, chemotherapy-free induction regimen (ie, oral arsenic and venetoclax) in NPM-mutated AML seems worthy of investigation in the future. This work was supported by grants from the National Natural Science Foundation of China (81970133 and 81820108004). The authors declare that they have no conflicts of interest.