BCL2 overexpression: clinical implication and biological insights in acute myeloid leukemia

Background BCL2 protein inhibitor venetoclax (ABT-199) has been authorized by Food and Drug Administration for relapsed/refractory chronic lymphoid leukemia with 17p deletion. Although venetoclax/ABT-199 also caused cell death in acute myeloid leukemia (AML), whether it could be applied to clinical treatment needs further studies. Here, we revealed clinical implication of BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using venetoclax. Methods BCL2 expression was analyzed in adult AML patients from public datasets The Cancer Genome Atlas (TCGA) and confirmed by another independent cohort from our own data. Results BCL2 expression showed up-regulated in AML patients among TCGA data and confirmed by our own data. BCL2 overexpression was correlated with FAB-M0/M1, whereas BCL2 under-expression was related to FAB-M5. However, BCL2 expression has no effect on overall survival (OS) and leukemia-free survival (LFS) of AML patients (determined in BCL2low and BCL2high groups). Interestingly, in the BCL2low group, patients undergoing autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-HSCT) had significantly better OS and LFS compared with patients only received chemotherapy, whereas, no significant difference was found in OS and LFS between chemotherapy and auto/allo-HSCT patients in the BCL2high group. BCL2 expression was found positively correlated with HOX family gene, and negatively correlated with tumor suppressor microRNA such as miR-195, miR-497, and miR-193b. Conclusions BCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and cytogenetically normal AML. Electronic supplementary material The online version of this article (10.1186/s13000-019-0841-1) contains supplementary material, which is available to authorized users.

midostaurin for AML with FLT3 mutations, which accounts for approximately 30% of AML patients [6]. Moreover, the approval of enasidenib, an IDH2 inhibitor, has also approved by FDA for IDH2-mutated AML as another breakthrough in AML therapy [7].
Located on chromosome 18q21.33, BCL2 gene is found in human B-cell lymphomas, which is first identified through cloning the breakpoint of a translocation of t (14;18) [8]. It has proven to be major negative regulator in apoptosis, playing key roles in neoplastic transformation and leukemogenesis [9]. BCL2 protein plays crucial role in inhibiting the influx of adenine nucleotides through the outer mitochondrial membrane, resulting in reducing ATP hydrolysis and inhibiting cytochrome-C release [10]. Based on its oncogenic role in cancer, a highly potent and selective inhibitor of BCL2, ABT-199, presents antitumor activity while sparing platelets [11]. In 2016, venetoclax (ABT-199) has been authorized by FDA for relapsed/refractory chronic lymphoid leukemia (CLL) with 17p deletion. Although ABT-199 also induced cell death in AML [12], whether it can be applied to clinical treatment needs further studies. Notably, the FDA granted accelerated approval to venetoclax in combination with hypomethylating agents azacitidine or decitabine or low-dose cytarabine for the treatment of newly-diagnosed AML in adults who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy [7]. Herein, we revealed clinical implication of BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using BCL2 inhibitor venetoclax.

Patients and ethics
A first cohort of 173 adult AML patients with BCL2 expression data from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/ and http://www.cbioportal.org/) were identified and included in this study [13]. A total of 73 patients accepted auto/allo-HSCT for consolidation treatment, and the remaining 100 patients only received chemotherapy. The main clinical and laboratory features of the AML patients were presented in Table 1. The study protocol was approved by the Washington University Human Studies Committee, and informed consents were obtained from all patients.
A second cohort of 154 AML patients and 35 healthy donors was also enrolled in the study. The main clinical and laboratory features of the AML patients were presented in Additional file 1. All participants provided informed consents, and the study was approved by the Institutional Review Board of the Affiliated People's Hospital of Jiangsu University.
Statistical analyses SPSS 22.0 and GraphPad Prism 5.0 were used for statistical analyses and figures creation. Mann-Whitney's U test was used for the comparison of continuous variables, whereas Pearson Chi-square analysis or Fisher exact test was applied for the comparison of categorical variables. The prognostic effect of BCL2 expression on leukemia-free survival (LFS) and overall survival (OS) was analyzed though Kaplan-Meier analysis using Logrank test. Univariate and multivariate proportional hazard regression analysis was performed using Cox regression.
The P value (two-tailed) equal or less than 0.05 in all statistical analyses was defined as statistically significant.

BCL2 overexpression in AML
A cohort of 173 de novo adult AML patients with BCL2 expression data from public TCGA datasets was used for differential expression analysis. By using the GEPIA (http://gepia.cancer-pku.cn/detail.php), we found BCL2 expression in AML patients was significantly increased compared with GTEx normal BM samples (P < 0.001, Fig. 1a). In order to confirm the results, we further analyzed BCL2 expression in the second cohort of 154 AML patients from our hospital. Similarly, BCL2 expression was markedly up-regulated in newly diagnosed AML compared with controls and AML patients achieved CR (P < 0.001 and = 0.041, Fig. 1b). Moreover, BCL2 transcript level was significantly increased in AML at relapse time compared with those at CR time (P = 0.024, Fig. 1b).

BCL2 expression identified specific FAB subtypes of AML
In order to explore the clinical implication of BCL2 expression in AML, we further divided these cases into two groups (BCL2 high and BCL2 low ) based on median level of BCL2 transcript. The comparison of clinical/laboratory characteristics of the AML patients between two groups were summarized in Table 1. There were no significant differences between BCL2 high and BCL2 low groups in sex, age, BM blasts, and the distributions of cytogenetics (P > 0.05). However, BCL2 high cases had significantly lower white blood cells (WBC) and higher peripheral blood (PB) blasts compared with BCL2 low cases (P = 0.041 and 0.033). Additionally, significant differences in the distributions of FAB classifications and cytogenetics were found between two groups (P = 0.000). BCL2 overexpression was markedly correlated with FAB-M0/M1 (P = 0.038 and 0.015), whereas BCL2 under-expression was associated with FAB-M5 (P = 0.001). Among gene mutations, no significant differences  were found, besides BCL2 high tended to be associated with WT1 mutations (P = 0.057).

BCL2 expression did not affect prognosis in AML
Among the tested AML patients, a total of 73 cases received auto/allo-HSCT for consolidation treatment (after induction chemotherapy), whereas the other 100 cases only received chemotherapy. In both chemotherapy and auto/allo-HSCT groups, BCL2 high patients showed similar OS (median 26.3 vs 15.8 months) and LFS (median 11.1 vs 9.3 months) time compared with BCL2 low patients ( Fig. 2a and c). Among cytogenetically normal AML (CN-AML), there was also no significant difference in OS (median 24.6 vs 18.1 months) and LFS (median 9.6 vs 11.6 months) time between BCL2 high and BCL2 low groups ( Fig. 2b and d). Moreover, no matter in either chemotherapy or auto/allo-HSCT groups, no significant differences were found in OS and LFS time between BCL2 low and BCL2 high groups among wholecohort-AML (Chemotherapy group: OS median 8.  (Fig. 2e-l). Moreover, Cox regression analysis also confirmed that BCL2 did not independently affect the OS and LFS in whole-cohort-AML (Table 2).

High expression of BCL2 in AML patients did not benefit from transplantation
To investigate whether AML patients with high expression of BCL2 could benefit from auto/allo-HSCT, survival in patients with auto/allo-HSCT were compared among both BCL2 high and BCL2 low groups. In the BCL2 low group, the patients undergoing auto/allo-HSCT had significantly better OS and LFS compared with patients only received chemotherapy among both total AML (OS median 56.3 vs 8.0 months and LFS median 13.8 vs 5.9 months) and CN-AML (OS median 56.3 vs 8.2 months and LFS median 13.8 vs 8.2 months) ( Fig. 3a-d).
In the BCL2 high group, no significant differences in OS and LFS were found between auto/allo-HSCT and chemotherapy groups among both total AML (OS median 30.0 vs 8.1 months and LFS median 14.6 vs 8.0 months) and CN-AML (OS median 24.6 vs 15.5 months and LFS median 12.0 vs 8.6 months) (Fig. 3e-h).

Discussion
In this study, we found and verified that BCL2 expression was significantly up-regulated in newly diagnosed AML especially in relapsed AML among two independent cohorts in consistent with previous studies [19][20][21][22][23][24][25][26][27][28]. Previously, BCL2 overexpression showed heterogenous expression in the range of 34 to 87% [19]. Although  clinical outcome of AML. Similarly, several investigators also did not show the significant association of BCL2 overexpression with prognosis [23,24]. In addition, increasing studies attempted to show the transcript ratio of FLT3 + KIT/BCL2, FLT3/BCL2, and BAX/BCL2 (or combined with WT1 or MDR1) may affect prognosis in AML [25][26][27][28]. Thus, we deduced that BCL2 expression was not a valuable single factor that affecting prognosis in AML. Apoptosis plays crucial roles in the command of tissue homeostasis, and is important in the clearance of infected, unwanted, or otherwise damaged cells [29]. Meanwhile, deregulation of apoptosis may give rise to neoplastic transformation [9]. It has been well demonstrated that BCL2 acted as a negative regulator on cellular apoptosis and is a druggable target [9,[30][31][32]. In hematologic malignancies, the impairment of apoptosis process is often caused by BCL2 overexpression [32]. Taking these into account, targeting BCL2 proteins to cause apoptosis is considered as a potential therapeutic approach in hematological malignancies [33][34][35][36]. Early efforts in BCL2 inhibitor including ABT-737 and ABT-263/navitoclax were encountered with disappointment in clinic because of dose-dependent thrombocytopenia [31]. In 2013, Souers et al. recently reported the reengineering of ABT-263/navitoclax to create ABT-199/ venetoclax, which was a highly potent and selective inhibitor of BCL2 [11]. By clinical studies, venetoclax presented high rate of treatment response as a single drugs in refractory/relapsed CLL [37]. Of note, ABT-199/venetoclax has been authorized by FDA for relapsed or refractory CLL with 17p deletion in 2016. In addition to CLL, ABT-199 also powerfully kills a various array of non-Hodgkin lymphoma and AML cell lines [12], suggesting that the drug has the potential to be efficacious in multiple hematologic malignancies. From our study, we observed that AML patients with BCL2 underexpression could benefit from auto/allo-HSCT, whereas patients with BCL2 overexpression did not benefit from auto/allo-HSCT.
Herein, we further determined the molecular signatures associated with BCL2 in AML to further get better understanding of AML biology. We found that BCL2 dysregulation was significantly associated with HOX gene family, which was reported highly correlated with hematopoiesis and leukemogenesis [38,39]. Moreover, for microRNAs, we found BCL2 expression was negatively correlated with several microRNAs such as miR-195, miR-497, miR-135a, miR-196a, miR-193b, miR-455, miR-375, and miR-205, which were found to be associated with AML pathogenesis or patients prognosis by previous studies [40][41][42][43][44]. Of these microRNAs, miR-195 and miR-497 was identified as predicted microRNAs that could direct target BCL2. Obviously, further studies are needed to confirm the direct connections of BCL2 with microRNAs by luciferase assay.

Conclusion
BCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and CN-AML.