Overexpression Of Bcl-2 Research Articles

Investigating the Dual Targeting of BCL-2 and CD38 in Models of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is correlated with poor prognosis and a high mortality. Current AML treatment often fails to achieve complete remission and relapse is common, highlighting the need for more targeted treatments. Overexpression of BCL2 is a hallmark of AML progression and is often associated with a poor response to cytotoxic treatment. BCL2 inhibition by the BH3 mimetic Venetoclax has been shown to be effective in promoting AML cell death and recently Venetoclax has received FDA approval for the treatment of AML. However, some patients do not respond or can develop resistance, therefore multiple studies for combinational therapies for Venetoclax have been researched. We have previously demonstrated CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from mesenchymal stromal cell (MSC) to AML blasts in the bone marrow (BM) microenvironment, which results in a reduction in AML derived oxidative phosphorylation and subsequent reduced leukemia growth and improved animal survival. We therefore investigated the consequences of inhibiting CD38 using daratumumab and BCL2 using Venetoclax on the AML survival. Primary AML blasts were isolated from patient's BM. CD38 expression and BCL2 expression was assessed by flow cytometry analysis of AML. AML had significantly higher BCL2 expression and a slight increase in CD38 expression compared to CD34+ cells. Venetoclax alone caused a significant decrease in cell viability, however daratumumab or in combination with Venetoclax had no additive effect on AML survival. Since AML is highly reliant on the BM microevironment we cultured AML on MSC with either Venetoclax alone, daratumumab alone, or Venetoclax and daratumumab for 24 hours. Cells were then stained with Annexin V-FITC/PI and analysed using flow cytometry. Cells underwent significantly more apoptosis in the combination Venetoclax and daratumumab treatment when compared to control AML cells. To determine the effect of Venetoclax and daratumumab treatment in preclinical models we used an NSG xenograft mouse model of AML, we transplanted MV411-luc or patient derived AML and treated the animals with either vehicle control (PBS) daratumumab (5mg/kg) on day 7 and 14 alone, Venetoclax (100mg/kg/day) alone, or both daratumumab and Venetoclax followed by bioluminescence imaging. In vivo, treatment with combination daratumumab and Venetoclax significantly reduced tumor burden and improved survival compared to control and either drug alone in the patient derived AML xenograft mouse model and in MV411. These data support the further clinical investigation of Venetoclax and Daratumumab combination as a therapeutic approach for the treatment AML. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

Neoantigens in Patients with De Novo Follicular Lymphoma: Results from the PRIMA Study

Neoantigens in Patients with De Novo Follicular Lymphoma: Results from the PRIMA Study

Combination of Homoharringtonine with Venetoclax and Azacitidine Excerts Better Treatment Response in Relapsed /Refractory Acute Myeloid Leukemia

Overexpression of Bcl-2 is known to be an important factor for drug resistance in relapsed /refractory acute myeloid leukemia (R/R-AML). Combination of Bcl-2 inhibitor venetoclax with hypomethylating agents (HMA) has shown promising killing effect in elder patients with newly diagnosed AML, also worked in R/R-AML but with low rate of complete response (CR). High expression of Bcl-XL and/or Mcl-1 is the main reason for resistance to venetoclax. As an inhibitor of both Bcl-XL and Mcl-1, homoharringtonine (HHT) has presented a synegetic effect with venetoclax in inhibition of Mcl-1 and killing of lymphama. In this study, we analyzed the treatment response and safety of combination of HHT (1mg/m2 d1-7) with venetoclax (400mg d1-14) and azacitidine (75mg/m2 d1-7) (HVA) in R/R-AML. From 6, 2019 to 5, 2020, in Nanfang Hopsital, 22 patients with RR-AML were treated with HVA (HVA group), with a median age of 39(16-62) years, previous chemotherapy of 3(1-7) cycles. 15 (68.2%) relapsed within one year after allogenetic hematopoietic stem cell transplantation (allo-HSCT) and 12 (54.5%) previously exposed to HMA treatment. Meanwhile, 7 patients with RR-AML were treated with venetoclax combined with HMA (control group), with a median of 42(31-74) years, previous chemotherapy of 3(1-6) cycles. 2 (28.6%) relapsed after allo-HSCT and 5 (71.4%) had HMA exposure. In HVA group, totally 11/17(64.7%) patients acquired treatment response, and 7/15(46.7%) obtained CR/CRi, of whom 6 presented minimal residual disease (MRD) negative. While in control group, only 2/7(28.6%) acquired treatment response and 1 obtained CR. Subgroup analysis in HVA group showed allover response rate (ORR) was 9/13(69.2%) and CR rate was 6/11(54.5%) (all with MRD negative) in the patients with allo-HSCT. And in the patients without allo-HSCT ORR was 2/4(50.0%) and CR rate was 1/4. With a median follow-up of 6(1-7) months, one patient was bridged to allo-HSCT, 2 out of 7 patients with CR relapsed. All patients were well tolorated to both treatment regimens, showing as a median neutropenia of 9.5(5-14) days, 5 patients with Febrile neutropenia and 4 with pulmonary infection. In conclusion, combination of HHT with venetoclax and azacitidine (HVA) excerts better treatment response in R/R-AML, especially in those with allo-HSCT, and shows well tolorated. A multi-center prospective clinical trial (NCT04424147) has been initiated. Key words: HVA regimen, relapsed /refractory acute myeloid leukemia, treatment response, safety Disclosures No relevant conflicts of interest to declare.

Impact of p53 and BCL2 Expression on Prognosis in Patients with Acute Myeloid Leukaemia

Introduction Acute myeloid leukaemia (AML) is a heterogenous disorder that arises from clonal expansion of malignant hematopoietic precursor cells. The tumor suppressor p53 gene plays an important role in regulation of the cell cycle and apoptosis. Somatic mutations of the p53 gene have been reported in 5-10% of all patients with AML, although the rate is higher in therapy-related disease and elderly patients. Alteration or loss of p53 is one of the most powerful independent indicators of poor outcome. BCL2 positivity in AML and its impact on prognosis is less well described. Methods This is a single-centre, retrospective analysis of AML patients treated over a ten year period (2006-2016). Patients were identified through our pathology database. P53 expression (as a surrogate marker for tp53 mutation) and BCL2 expression were analysed by immunohistochemical (IHC) analysis of marrow biopsies (greater than 30% was considered positive). We analyzed AML presentation, management and overall survival (OS), and correlated these with presence of p53 mutation and BCL2 expression. Results We identified 48 patients; the majority were elderly (median age 68.5 years). Thirty-four patients (71%) haddenovopresentation of AML, 6 had underlying Myelodysplastic Syndrome (13%), 4(8%) progressed to AML from underlying Myelofibrosis, and 8% had therapy-associated AML. Patient and disease characteristics are outlined inTable 1. Median blast count by IHC of bone marrow biopsies was 70%. The results of cytogenetic analysis were available for 38 patients (79%). Fourteen patients (37%) had normal cytogenetics, but a wide range of genetic abnormalities were identified(Table 1). NPM1 (for 22 patients) and FLT3 mutation analysis (for 33 patients) were available, and reported as positive in 4(18%) and 5(15%) respectively. p53 overexpression was identified in 6 patients (12.5%). 50% of these had secondary AML, and the remainder weredenovopresentations. Four of these patients (66%) had complex cytogenetics, one had deletion 5q and cytogenetic analysis was not available for the remaining patient. Median age of patients who were p53 positive was 60 years, compared to 70 years in those without p53 overexpression. BCL2 expression analysis showed a median value of 60% (range 5-100%). BCL2 positivity was seen in 39 patients (81.2%); half of this cohort (15 patients) had normal cytogenetics and only five patients (16%) had abnormalities that indicated poor risk cytogenetics (complex, deletion 7q, etc.). Thirty-nine patients (83%) received active treatment and nine received supportive care only due either to frailty or patient choice. Twenty-two patients (60%) had induction chemotherapy, 7(19%) received Azacitadine, and a further 7 patients had low-dose cytarabine. Nineteen patients (51%) had relapsed/refractory disease. Five patients (13.5%) with BCL2 positive disease received Venetoclax as a single agent for relapsed or refractory disease. Median OS was 11.5 months (range 0-72); 39 patients (81%) had died at the time of analysis. 26 died from disease progression (79%), and 7(19%) of sepsis. One patient was lost to follow up. OS in those with wild-type p53 was 11.5 months, which compared favorably to 7.5 months in those with p53 mutation. BCL2 overexpression also conferred a poorer prognosis; median OS was 10 months compared to 17 months in BCL2 negative disease. Conclusion This study reflects the potential use of p53 and BCL2 in real-life practice in assessing prognosis in AML patients outside of a clinical trial setting. We confirmed the previously reported poor prognosis of p53 alterations in AML (OS 7.5 months), and the study also indicates a poorer prognosis in patients with BCL2 positivity. The widespread availability of IHC techniques for trephine biopsies and the potential for rapid turn-around times in routine clinical laboratories suggests that further studies of the prognostic impact of such gene alterations are warranted. Disclosures No relevant conflicts of interest to declare.

Bclw Overexpression Predicts Aggressive Disease in B-Cell Lymphomas

B-cell lymphomas encompass a phenotypically and genetically heterogenous subgroup within hematologic malignancies. Despite this heterogeneity, the ability to evade apoptosis is a unifying feature across B-cell lymphomas, and alterations within the Bcl-2 family of apoptosis regulatory proteins is a key mechanism for this evasion. While overexpression of the anti-apoptotic Bcl-2 family member BCL2 has been widely described in multiple B-cell lymphomas, altered expression of other anti-apoptotic proteins within this family, including BCLX, MCL1, A1 and, in particular, BCLW has been under-investigated. This is highlighted by clinical trials of the BCL2 inhibitor, venetoclax, which found poor single agent activity in B-cell lymphomas that were thought to be highly BCL2-dependent. Therefore, we investigated the contributions of anti-apoptotic Bcl-2 proteins in B-cell lymphomas. Analysis of gene expression profiling datasets of patient samples of major B-cell lymphoma subtypes as compared to normal B-cells demonstrated differential overexpression of anti-apoptotic Bcl-2 proteins, with BCL2, BCLX and BCLW being significantly overexpressed in multiple B-cell lymphoma subtypes. Focusing on aggressive B-cell lymphoma subtypes, BCLW was consistently overexpressed across all major B-cell lymphoma subtypes. Notably, BCLW overexpression was maintained in high grade follicular lymphoma compared to low grade, whereas BCL2 levels decreased as grade increased. These findings indicate that BCLW overexpression is selected for in more aggressive B-cell lymphoma subtypes. BCLW overexpression in aggressive B-cell lymphoma cell lines conferred resistance to the non-specific BH3 mimetic navitoclax as well as to venetoclax. Further, analysis of diffuse large B-cell lymphoma (DLBCL) cell lines demonstrated that the combination of high BCLW/low BCL2 in GCB DLBCL and high BCLW/moderate BCL2 in non-GCB DLBCL is also associated with decreased navitoclax sensitivity, suggesting that high BCLW may confer treatment resistance. In accordance with this, analysis of patient outcomes in DLBCL demonstrates that patients exhibiting high BCLW levels with low BCL2 levels have poorer survival than those with low BCLW. Moreover, Cox regression analysis showed BCLW was an independent determinant of overall survival in DLBCL. Together, our results indicate that BCLW predicts for more aggressive disease in B-cell lymphomas and increased resistance to therapy. Disclosures Eischen: AbbVie Inc.:Research Funding.

A Novel BCL2-Driven Compound Mutant Mouse Model for In Vivo Research on BH3 Mimetics

Introduction: Eµ-TCL1-transgenic mouse models are often applied to discover and observe the development and kinetic of chronic lymphocytic leukaemia (CLL), as they develop diseases most similar to human CLL with a very high penetrance. To gain a better understanding on new therapy options and their effect on disease regression it is very important to observe therapy response, overall survival and symptoms during treatment of the disease not only in vitro but also in vivo in a suitable mouse model. However, application of BH3 mimetics like venetoclax is limited in the classical Eµ-TCL1 mouse model, since these mice are resistant towards venetoclax treatment. Therefore, we have generated a novel mouse model with Eµ-TCL1 as back bone and conditional overexpression of BCL2. Methods and results: We established a new mouse model (TBC) by crossbreeding mice expressing Eµ-TCL1tg/wtwith mice containing a B-cell specific conditional Bcl-2Rosa26/wt; Cd19CreCre/wtoverexpression and compared the disease kinetics to classical Eµ-TCL1 mice and to BC mice. TBC animals exhibit a severe leukocytosis at very early stages of disease development (12 weeks; mean 96.000/µl) in comparison to TC (15.100/µl) and BC (81.900/µl) mice. TBC mice develop CD23low/CD21neg leukemic B cells as they are known from TC mice with CD19+/CD5+ expression. Indeed, these mice show a significantly shortened overall survival of ~300 days (n=43) compared to TC mice (n=106; ~350 days; p<0.001) and BC mice (n=28; ~410 days; p<0.001) with severe clinical symptoms such as splenomegaly and cachexia. Strikingly, in contrast classical TC mice, which are resistant towards venetoclax, isolated B-cells of TBC mice are 10-times more sensitive towards venetoclax in vitro (0,02 µM) and can also be killed by the MCL1 inhibitors in nanomolar ranges, but not by BCL-xl inhibitors (>2µM). Based on our in vitro data, we have treated TBC mice with venetoclax and observed an early and dramatic drop of leukocytes to normal ranges within the first two weeks of treatment. Leukocyte reduction lasted for the whole period of treatment. When investigating the spleens after sacrificing the mice they showed high amounts of dead cells inside the spleens, indicating that venetoclax was also efficient in lymphatic tissues as we know it from human trials. Conclusions: Autochthonous mouse models on which BH3 mimetics can be tested are rare. In our mouse model apoptosis screening in vitro we can show good results for BH3 mimetics with a high sensitivity already in low dosing. The BCL2-driven TCL1 mouse model enables the investigation of treatment with venetoclax in vivo to gain a better understanding of this frequently on patients applied therapy. Moreover, this model will help us to test other drugs (like MCL1 inhibitors) in combination with venetoclax to identify synergistic drugs in vivo in a timely manner. Furthermore, this model will offer us the opportunity to identify treatment strategies to overcome venetoclax resistance in vivo. Disclosures No relevant conflicts of interest to declare.

A Phase 1b-2 Study of KRT-232, a First-in-Class, Oral, Small Molecule Inhibitor of Murine Double Minute 2 (MDM2), in Combination with Acalabrutinib for the Treatment of Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) or R/R Diffuse Large B-Cell Lymphoma (DLBCL)

A Phase 1b-2 Study of KRT-232, a First-in-Class, Oral, Small Molecule Inhibitor of Murine Double Minute 2 (MDM2), in Combination with Acalabrutinib for the Treatment of Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) or R/R Diffuse Large B-Cell Lymphoma (DLBCL)

Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

Combination of cancer-specific prodrug nanoparticle with Bcl-2 inhibitor to overcome acquired drug resistance

Multiple combination therapies with chemotherapeutic drugs and inhibitors of drug resistance have been effective in the clinical cases, but concerns have been raised about the severe toxicity of these chemotherapeutic drugs. Herein, we report a potent and safe combination strategy of cancer-specific doxorubicin (DOX) prodrug nanoparticles (PNPs) and B-cell lymphoma-2 (Bcl-2) anti-apoptotic inhibitor, Navitoclax, to overcome acquired drug resistance during chemotherapy. The cancer-specific PNPs were constructed by conjugating cathepsin B-specific cleavable peptide (Phe-Arg-Arg-Gly; FRRG) to DOX, resulting in FRRG-DOX that self-assembled into nanoparticles and the FRRG-DOX nanoparticles were further stabilized with the FDA-approved pharmaceutical excipient, Pluronic F68. The resulting PNPs are specifically cleaved and metabolized to free DOX in cathepsin B-overexpressing cancer cells, but they exhibited minimal cytotoxicity in cathepsin B-deficient normal cells. As expected, free DOX and PNPs induced overexpression of Bcl-2 in MDA-MB-231 cells, due to acquired drug resistance in a cell culture system. However, combination therapy with PNPs and Navitoclax showed the outstanding synergetic cytotoxicity by decreasing the expression level of Bcl-2. In MDA-MB231 breast tumor-bearing mice, intravenously injected PNPs efficiently accumulated in targeted tumor tissues via enhanced permeability and retention (EPR) effect. When combined with orally administered Navitoclax, PNPs exhibited more potent therapeutic efficacy in aquired drug resistant models than free DOX plus Navitoclax, whereas PNPs greatly reduced systemic toxic side effects in normal organs. Our cancer-specific PNP-based combination therapy with Bcl-2 inhibitor may provide a promising approach for the potent and safe treatment of acquired drug-resistant cancers.

Abstract PO-05: TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center (GC) B cells. Despite progress in the treatment of DLBCL, approximately 40% of patients relapse or are refractory to the treatment, which usually leads to fatality. DLBCLs are characterized by aberrant DNA methylation and this feature correlates with poor clinical outcome. We and others have shown that deregulated epigenetic mechanisms contribute to lymphoma formation. Particularly, we have demonstrated that TET2, an enzyme that converts methylcytosine (mC) into hydroxymethylcytosine (hmC) and is mutated in ~10% of DLBCLs, is a B-cell tumor suppressor. GC-specific deletion of TET2 (Cg1Cre/Tet2-/-) resulted in accelerated lymphomagenesis in DLBCL mouse models driven by BCL6 overexpression, with 100% Cg1Cre/Tet2-/-;ImBcl6 mice developing lymphoma at 7 months compared to only 50% in ImBcl6 control mice. In addition, TET2 deletion in hematopoietic stem cells (VavCre/Tet2-/-) induced GC B-cell hyperplasia (B220+GL7+CD95+ cells; 10% VavCre/Tet2-/- vs 5% VavCre/Tet2+/+), promoting malignant transformation. Further analysis of the GC reaction revealed that TET2-deficient GC B cells displayed anomalous patterns of DNA methylation. GC B cells from VavCre/Tet2-/- mice presented 1) focal loss of hmC—using hMeDIPseq—with 25,000 differentially hydroxymethylated regions (DHMR) lost compared to VavCre/Tet2+/+ GC B cells and 2) increased mC—using RRBS—with almost 11,000 differentially methylated cytosines (DMCs), 84% hypermethylated, compared to VavCre/Tet2+/+ GC B cells. TET2-mediated reduction of hmC and hypermethylation affected enhancers and promoters, respectively, of genes mediating GC exit and terminal differentiation of GC B cells, especially those regulated at enhancers by the opposing functions of CREBBP and HDAC3. We are currently investigating the potential cooperative role between TET2-mediated hmC and CREBBP-mediated H3K27Ac, supported by reduced H3K27Ac at enhancers activated by CREBBP in VavCre/Tet2-/- GC B cells and mutual exclusion between TET2-mutant and CREBBP-mutant primary DLBCL. RNA sequencing analysis revealed that the genes epigenetically regulated by TET2 were aberrantly repressed in VavCre/Tet2+/+ GC B cells, explaining the observed GC hyperplasia in TET2-deficient GC B cells since these genes control the differentiation of GC B cells into plasma cells. Importantly, TET2-mutant DLBCL primary samples display a similar repressive transcriptional signature associated with GC B-cell terminal differentiation. Our data show how TET2-induced epigenetic changes contribute to lymphoma development and highlight the multilayered nature of the epigenome, which can be therapeutically exploited. We are evaluating the therapeutic potential in TET2-mutant DLBCL of a combinatorial therapy consisting of DNA methylation inhibitors (DNMTi), to revert hypermethylation at promoters, plus specific HDAC3 inhibitors, to compensate for the loss of hmC at enhancers. Citation Format: Pilar M. Dominguez, Wojciech Rosikiewicz, Xiaowen Chen, Hussein Ghamlouch, Said Aoufouchi, Olivier A. Bernard, Ari M. Melnick, Sheng Li, Ricky W. Johnstone. TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-05.

Decreased BIM expression in BCL2-negative follicular lymphoma: a potential mechanism for resistance to apoptosis

Follicular lymphoma (FL) is characterized by t(14; 18)(q32; q21), leading to overexpression of the antiapoptotic molecule BCL2; however, a subset of FLs lack BCL2 rearrangement and BCL2 expression as analyzed by immunohistochemistry (IHC). In this study, we evaluated expression of antiapoptotic (MCL1 and BCL-XL) and proapoptotic proteins (BIM) by IHC in both BCL2(-) and BCL2(+) FLs. FLs diagnosed between 2009 and 2019 were reviewed to identify BCL2(-) cases by IHC (assessed by clone 124). Immunohistochemical analyses for BCL2 (EP36), MCL1, BIM, BCL-XL, and Ki-67 were performed on tissue microarrays or whole slides. BCL2 (EP36) was interpreted as positive (≥10%) or negative (<10%). Ki-67 was interpreted on tumor cells in 10% increments. The remaining immunohistochemical analysis results were scored on tumor cells in 10% increments, and intensity was interpreted as weak, moderate, or strong to derive an H-score. Twenty-four BCL2(-) FLs were initially identified, but on further testing with BCL2(EP36) immunohistochemical staining, 5 of 24 were reclassified as BCL2(+), leaving 19 BCL2(-) FLs. Thirty-three BCL2(+) FLs were selected with sufficient tissue for additional immunohistochemical analyses. There was no significant difference in expression of antiapoptotic BCL-XL or MCL1 between BCL2(-) and BCL2(+) FLs (p=0.75 and 0.28, respectively). However, proapoptotic BIM expression was significantly lower in BCL2(-) FLs than in BCL2(+) FLs (p=0.002). In our study, 21% of putative BCL2(-) FLs were BCL2(+) when tested with alternative clones, supporting the practice of having more than one BCL2 clone in immunohistochemical laboratories. Decreased BIM expression in BCL2(-) FLs could have an overall antiapoptotic effect and represent an alternate mechanism for cell survival in BCL2(-) FLs.

CYTOTOXICITY OF FIVE PLANT EXTRACTS AGAINST DIFFERENT HUMAN CANCER CELL LINES AND THEIR MOLECULAR MECHANISM

Objective: Five methanol plant extracts namely, Euphorbia hierosolymitana, Dracaena marginata, Cordyline terminalis, and Sapium sebifera were screened in vitro for their cytotoxic effect on human tumor cell lines; namely, PC-3, HepG-2, HCT-116, and MCF-7 (prostate, liver, colon, and breast cancer) and human normal cell lines, namely, BJ-1.&#x0D; Methods: E. hierosolymitana (extract A) selectively inhibited HCT-116 cell proliferation with IC50 of 4.22 μg/ml, both Dracaena marginata (extract B) (bark, leaves, and branches) showed moderate cytotoxicity on MCF-7 with IC50 of 22.4 and 48.2 μg/ml, respectively. The remaining two extracts showed minimal to insignificant percentage inhibition on all cell lines.&#x0D; Results: E. hierosolymitana extract was selected for further studying, where the effect of the extract on the HCT116 cells showed a downregulation of her2 and Bcl-2 gene and overexpression of the Bax gene. Flow cytometry analysis was also performed to understand the effect of E. hierosolymitana on the apoptosis induction and the cell cycle. The results showed the induction of early and late apoptosis by 5.81 and 10.01%, respectively.&#x0D; Conclusion: Our results infer that E. hierosolymitana has a promising chance in fighting colorectal cancer due to its high cytotoxic effects on HCT116 cancer cells while having minimal effects on the BJ-1 normal cell lines.

Prognostic significance of Bcl-2 expression in primary cutaneous B-cell lymphoma: a reappraisal

Bcl-2 family protein plays an important role in apoptosis and its overexpression is protects neoplastic cell from apoptotic stimuli. Cutaneous B-cell lymphoma are rare non-Hodgkin lymphomas and can be classified in primary forms, featuring an exclusive skin-involvement at diagnosis, and cutaneous spread of a nodal disease. Such a distinction is not trivial, owing to different prognosis (indolent vs. aggressive) and therapeutic management. Bcl-2 expression at immunohistochemistry can be crucial in differential diagnosis between cutaneous and systemic disease, as well as between the different primary cutaneous forms. In the last few years, an animated debate on the prognostic role of Bcl-2 overexpression at molecular analysis have been developed in cutaneous B-cell lymphoma. To conclude, Bcl-2 expression have a diagnostic role more than prognostic in primary cutaneous B-cell lymphomas.

Lymphome du manteau orbitaire traité avec succès par un inhibiteur de Bcl-2 : à propos d’un cas

Lymphoma is the most common orbital malignancy in adults. Among the types of lymphoma, mantle cell lymphoma is a particularly aggressive form, often discovered through systemic involvement, with a dismal prognosis due to frequent recurrences. It is secondary to a t (11; 14) (q13; q32) chromosomal translocation resulting in an anti-apoptotic signal via overexpression of Bcl-2. Treatment is based on R-CHOP poly-chemotherapy. We describe the case of a patient with an orbital recurrence of mantle cell lymphoma successfully treated with oral Bcl-2 inhibitor monotherapy. A 58-year-old man who was treated with R-CHOP 8 years ago for mantle cell lymphoma, in remission for 5 years, presented with progressive decreased visual acuity in the left eye, along with binocular diplopia. Clinical examination revealed a decrease in visual acuity in the left eye to 1/20 Parinaud 20 and a relative afferent pupillary defect on the left. External examination revealed a left cranial nerve VI palsy, 2mm of painless proptosis, and hypesthesia of the left V1 territory, leading to a diagnosis of left orbital apex syndrome. The disc and macular OCT were normal. The visual field showed enlargement of the left blind spot. An emergency CT scan and MRI revealed an apical extraconal tissue mass infiltrating the medial rectus muscle, extending to the superior orbital fissure, optic canal and left cavernous sinus, hyperintense on T2 weighted images and isointense on T1. The morphological appearance was strongly suggestive of an infiltrative lymphomatous process. An 18 FDG PET-scan identified the orbital lesion as well as enhancing lesions in the axilla and colon; given the clinical features and test results, the diagnosis of recurrent mantle cell lymphoma was made without biopsy. Treatment with Venetoclax (Bcl-2 inhibitor) was initiated. At one month of treatment, the orbital apex syndrome had entirely resolved, with visual acuity increased to 8/10 Parinaud 4 and a metabolic return to normal on PET scan. The PET scanner and clinical examination at 3 months were entirely normal. At the one-year follow-up visit, the patient was still on Venetoclax, the clinical examination was unchanged, and the PET-scan still showed a complete metabolic response.

The temporal effects of topical NF-κB inhibition, in the in vivo prevention of bile-related oncogenic mRNA and miRNA phenotypes in murine hypopharyngeal mucosa: a preclinical model.

Supraesophageal bile reflux at strongly acidic pH can cause hypopharyngeal squamous cell cancer, through activation of the oncogenic NF-κB-related pathway. We hypothesize that topical pre- or post-application of pharmacologic NF-κB inhibitor, BAY 11-7082 (0.25 μmol), on murine (C57BL/6J) HM (twice a day for 10 days) can effectively inhibit acidic bile (10 mmol/l; pH 3.0) induced oncogenic molecular events, similar to prior in vitro findings. We demonstrate that the administration of BAY 11-7082, either before or after acidic bile, eliminates NF-κB activation, prevents overexpression of Bcl2, Rela, Stat3, Egfr, Tnf, Wnt5a, and deregulations of miR-192, miR-504, linked to bile reflux-related hypopharyngeal cancer. Pre- but not post-application of NF-κB inhibitor, significantly blocks overexpression of Il6 and prostaglandin H synthases 2 (Ptgs2), and reverses miR-21, miR-155, miR-99a phenotypes, supporting its early bile-induced pro-inflammatory effect. We thus provide novel evidence that topical administration of a pharmacological NF-κB inhibitor, either before or after acidic bile exposure can successfully prevent its oncogenic mRNA and miRNA phenotypes in HM, supporting the observation that co-administration of NF-κB inhibitor may not be essential in preventing early bile-related oncogenic events and encouraging a capacity for further translational exploration.

MiR-202 Suppresses Hepatocellular Carcinoma Progression via Downregulating BCL2 Expression.

miRNAs play an important role in progression of hepatocellular carcinoma (HCC). In this work, we assessed the function of miR-202 in human HCC and identified BCL2 as its target. We found miR-202 expression was found significantly downregulated, while BCL2 expression was markedly upregulated in HCC tissues and cell lines (HepG2, Hep3B, and HCCLM3). Both miR-202 and BCL2 were closely correlated with major vascular invasion and advanced TNM stage as well as overall survival of HCC patients. Overexpression of miR-202 significantly inhibited cell proliferation, induced apoptosis and cell cycle arrest at the G0/G1 phase, and prevented tumor formation in a xenograft nude mouse model. Further, miR-202 dramatically inhibited migration, invasion, and epithelial–mesenchymal transition. miR-202 bound to the 3′-untranslated region (3′-UTR) of BCL2 mRNA and downregulated the expression level of BCL2 protein. Exogenous BCL2 overexpression weakened the inhibitory effects of miR-202, while inhibition of BCL2 enhanced the inhibitory effects of miR-202. In conclusion, miR-202 serves as a tumor suppressor in HCC progression by downregulating BCL2 expression, indicating miR-202 might be a potential target for HCC.

Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells.

Post-implantation cell survival and angio-/vasculogenesis are critical for the success of cell-based regenerative strategies. The current study aimed to overexpress B-cell lymphoma 2 (Bcl-2) gene in dental pulp stem cells (DPSCs) and examine the anti-apoptotic and angio-/vasculogenic effects both in-vitro and in-vivo. DPSCs were transduced with Bcl-2-green fluorescent protein (GFP) lentiviral particles and examined for cell proliferation and apoptosis. The cells were cultured under normoxic or hypoxic (0.5 mM CoCl2) conditions and examined for the expression of angiogenic factors and effects on endothelial cell proliferation, migration and vessel morphogenesis. Cells with or without hypoxic preconditioning were used in in-vivo Matrigel plug assay to study the post-implantation cell survival and angio-/vasculogenesis. Bcl-2-overexpressing-DPSCs showed significantly lower apoptosis than that of null-GFP-DPSCs under serum-free conditions. Under hypoxia, Bcl-2-overexpressing-DPSCs expressed significantly higher levels of vascular endothelial growth factor compared to that under normoxia and null-GFP-DPSCs. Consequently, Bcl-2-overexpressing-DPSCs significantly enhanced endothelial cell proliferation, migration and vascular tube formation on Matrigel. Immunohistological assessment of in-vivo transplanted Matrigel plugs showed significantly higher cell survival and vasculature in hypoxic preconditioned Bcl-2-overexpressing-DPSC group compared to null-GFP-DPSC group. In conclusion, Bcl-2 overexpression and hypoxic-preconditioning could be synergistically used to enhance post-implantation cell survival and angio-/vasculogenic properties of DPSCs.

Differential mechanisms involved in RG-7388 and Nutlin-3 induced cell death in SJSA-1 osteosarcoma cells

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

Abstract 4741: Exploring possible drug resistance and sensitivity mechanisms in treatment-refracotry betel-nuts related HNSCC cell line(TW2.6) by whole exon sequencing and molecular signaling for future drug combinations

Abstract Background: Betel nut chewing might contribute to (1)strong inflammation, angiogenesis, &amp; invasion; (2)resistance to traditional therapies. Betel-nuts related HNSCC in Taiwan might belong to mesenchymal differentiation and have the worst prognosis. Our group found betel-nuts exposed HNSCC cell line, TW2.6, was resistant to traditional therapies. PDL1 western blotting was strong over TW2.6 with defective p53 mutation, p16 loss, and BCL2 overexpression. In our previous studies, Astragalus polysaccharides, PI3Kalpha inhibitor, AKT inhibitor, FGFR inhibitor, ALK/IGF1R inhibitor, CDK4/6 inhibitor, BCl2 inhibitor, WEE1 inhibitor, ATR inhibitor, DNA-PK inhibitor, &amp; VEGFR2/ PDGFR/FGFR or VEGFR2/c-MET/Axl triple blockage might be effective on TW2.6 and reverse treatment refractoriness, maybe through the inhibition of mesenchymal transformation, pRB, &amp; PI3K/AKT /mTOR signaling and the modulation of stemness &amp; PD1/PDL1 pathway. Afatinib &amp; CDK4/6 inhibitors also have immuno-modulatory effects in our studies. Purpose &amp; Methods: NGS studies will be introduced to representative HNSCC cell lines to study molecular phenotypes of these HNSCC cell lines (including SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, &amp; TW2.6) for future drug combinations &amp; basic/translational research. Results: TW2.6 had PIK3CA H1047R mutation, high TMB(8.42 muts/Mb)/MSS, and VEGF-A amplification. We found afatinib response on several cell lines not so correlated to EGFR expression &amp; CNV status. Palbociclib response seemed to correlate to CCND1 gain and p16 loss; but FaDu had not so good palbociclib response even with these two changes and TW2.6 has good response even without these two. TW2.6 also had other genomic changes, such as HRAS &amp; DDR2 mutations, FAT1 loss, amplification of FGF10/CCND3/SOX9, and deletions of STK11/ARID1B/TNFAIP3. Conclusions: PI3K inhibitor, anti-angiogenesis therapies, and immunotherapy-containing regimens will be future combination options for betel-nuts related HNSCC by TW2.6 molecular signatures. Afatinib, CDK4/6 inhibitors, or DDR interventions could also enhance ICIs efficacy. The genomic landscapes of TW2.6 also deserve further studies about CDK4/6 inhibitor resistance, immunotherapy resistance, epigenetic modifications, novel targeted therapy, &amp; biomarkers exploration. Mutations in TW2.6TW2.6GenesN=1%TP531100%SPEN1100%FAT11100%MUC61100%BUB1B1100%EPHB11100%HRAS1100%PTPRD1100%ATM1100%CDK121100%CDK81100%DDR21100%GNAS1100%MYC1100%PDGFRB1100%PIK3CA1100%TNFSF111100%TSC21100% Citation Format: Jo-Pai Chen, Jui-Ying Chang, Ruey-Long Hong. Exploring possible drug resistance and sensitivity mechanisms in treatment-refracotry betel-nuts related HNSCC cell line(TW2.6) by whole exon sequencing and molecular signaling for future drug combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4741.

Crosstalk between Noxa, Bcl-2, and ceramide in mediating p53-dependent apoptosis in Molt-4 human T-cell leukemia.

Ionizing radiation induces apoptosis in human Molt-4 leukemia cells in a p53-dependent manner. The tumor suppressor p53 stimulates various downstream targets that presumably trigger, individually or in concert, de novo ceramide synthesis and intrinsic apoptosis via mitochondrial outer membrane permeabilization (MOMP). Among these targets, BH3-only protein Noxa was found to be promptly activated by p53 prior to ceramide accumulation and apoptosis in response to irradiation. To evaluate the relation between Noxa and ceramide in irradiation-induced apoptosis, Noxa was silenced in Molt-4 cells and apoptosis, p53 expression, and ceramide accumulation were assessed in response to irradiation. In the absence of Noxa, irradiation of Molt-4 cells still induced apoptosis in a p53-dependent manner however ceramide levels decreased significantly although they remained higher than untreated control. Upon irradiation, Noxa was found to translocate to the mitochondria where endogenous ceramide accumulation was observed. In contrast, overexpression of Bcl-2, another mitochondrial protein, in Molt-4 cells abolished the endogenous ceramide accumulation and apoptosis. In irradiation-induced, p53-dependent pathways of apoptosis, the pro-apoptotic Noxa represents one of several, yet to be identified, pathways simultaneously triggered by p53 to produce mitochondrial ceramide accumulation and apoptosis. In contrast, Bcl-2 functions as a broader inhibitor of both ceramide accumulation and apoptosis. Altogether, these results indicate that members of the Bcl-2 family differentially regulate ceramide accumulation and reveal the existence of crosstalk between Bcl-2 family members and ceramide in mediating p53-dependent apoptosis in Molt-4 human T-cell leukemia.