Pro-survival Proteins Research Articles

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

SDC2 Expression Is Increased in Myeloma Cells in Response to Loss of Pro-Survival Surface Proteins, CD28 and CD86

Introduction: While recent advances in multiple myeloma (MM) have improved overall survival, most patients will succumb to late-stage and drug resistant disease. Plasma cell (PC) survival outside of the bone marrow is characteristic of late-stage myeloma and pivotal to disease progression. PCs are typically restricted to the bone marrow microenvironment, as they are dependent on stromal interactions for survival. One such interaction is the engagement of CD28 and its ligand CD86, proteins which are best understood for their roles in T-cell activation by antigen presenting cells. However, myeloma cells can express both CD28 and CD86 on the cell surface. Notably, high CD28 expression is seen in 59% of medullary recurrences, and over 90% of extramedullary relapses. Moreover, high CD86 expression is more common in malignant plasma cells as compared to precursor stages and has a reported role in driving expression of IRF4. We have previously shown that expression of CD28 and CD86 is associated with worse outcomes than that observed in patients with low expression in the MMRF CoMMpass study (NCT01454297). We have also shown that silencing of CD28 or CD86 results in cell death and loss of IRF4 protein expression in human myeloma cell lines (HMCL). We confirmed these studies using CRISPR/Cas9 editing and demonstrated loss of proliferation and induction of cell death in HMCLs. However, the mechanism(s) by which CD28 and CD86 contribute to MM cell survival is not fully understood. Methods: To shed light on the mechanistic basis of CD28 and CD86 in the survival of MM, we used CRISPR/Cas9 editing to disrupt expression from RPMI8226 myeloma cells. We then passaged transduced cells to select for populations that can survive loss of CD28 or CD86. These passaged cells were sorted into populations that either retained CD28 or CD86 expression as transduction controls (High), or populations that underwent CRISPR/Cas9 depletion (Low). Surface expression of CD28 High/Low and CD86 High/Low populations was confirmed via flow cytometry and were then processed for RNA isolation and cDNA library preparation. RNA-sequencing was performed using paired-end sequencing, and data were aligned using the STAR aligner. Differentially expressed genes were determined using edgeR with an FDR ≤ 0.05 and a fold-change ≥ 2.0. Validation of RNA-sequencing results was further assessed via western blot and flow cytometry. Results: Analysis of RNA-sequencing results indicated that 57 genes were differentially expressed between High and Low cells, with 23 genes common to both CD28 and CD86 High/Low comparisons. (FDR<0.05). Notably, our results reveal that loss of CD28 or CD86 is associated with an increase in mRNA expression of Syndecan-2 (SDC2). In addition, our preliminary experiments evaluating SDC2 surface expression via flow cytometry suggest that both CD28 Low and CD86 Low populations have increased expression of SDC2. Additionally, in the RNA-seq analysis we also observed that CD28 Low and CD86 Low cells were also able to sustain mRNA expression of IRF4. This result was later confirmed via western blot, demonstrating CD28 Low and CD86 Low cells sustain protein expression of IRF4. Conclusions: Our results demonstrate that RPMI8226 cells that lack CD28 or CD86 expression have increased mRNA and protein expression of SDC2, a member the heparan sulfate proteoglycan family. Since SDC2 expression has also been associated with a number of cancers including colorectal, lung, and pancreatic cancers, these results provide insight to potential alternative pathways that contribute to myeloma survival independent of CD28 and CD86 signaling. We also confirmed that CD28- and CD86-depleted populations were able to maintain IRF4 mRNA/protein levels. This indicates myeloma cells have mechanisms to sustain IRF4 expression in addition to CD28 and CD86 signaling. These results are also consistent with previous findings illustrating importance of IRF4 in myeloma. Finally, we have developed CRISPR/Cas9-depleted CD28 and CD86 populations in additional HMCLs. We are currently in the process of isolating CD28 High/Low and CD86 High/Low populations for additional analysis and will present these upcoming results.

Significance of the Apoptotic Regulators BAX, BCL2, BCL- XL and MCL1 in Newly Diagnosed AML

Introduction: In acute myeloid leukemia (AML), cytogenetics and molecular mutations remain important prognostic tools for tailoring treatment after induction therapy. Abnormalities in multiple components of apoptotic pathways have been identified as potential drivers of AML. Identifying other biomarkers may help us further refine prognosis and identify other therapeutic strategies. B-cell lymphoma 2 (Bcl2), Bcl-xL and myeloid cell leukemia-1 (Mcl1) are commonly expressed prosurvival proteins in hematologic malignancies, whereas Bax acts as a promoter of apoptosis. We retrospectively evaluated the significance of Bax, Bcl2, Bcl-xL and Mcl1 expression in newly diagnosed AML patients using the Beat AML program 1. Methods: We analyzed RNA-expression data from The Beat AML study (672 tumor specimens collected from 562 patients) 1. Data was extracted from the public database. Different expression levels of the above genes were compared using t-tests. Results were considered significant with p-value < .05. Kaplan-Meier curves were used to estimate survival. Results: For the correlative survival analyses, Bax, Bcl2, Bcl-xL and Mcl1 mRNA expression and clinical information were both obtained from the BeatAML cohort 1. Patients were categorized into high expressers (≥ median) and low expressers (<median). In total, 461 patients had available gene expression data. The mean age of diagnosis was 57 years, and 56.6% of patients were male. The most common specific diagnosis at inclusion was AML with mutated NPM1 (22%), followed by AML with myelodysplasia related changes (21%) and AML, NOS (11%). Complete response to induction was seen in more than half (56%) of the patients for which response was recorded (n=369). Others developed refractory AML (32%). No data was available for 12%. Standard chemotherapy (induction therapy with idarubicin and cytarabine (7+3) and consolidation therapy with high dose cytarabine) was used in 98.4% of these patients. Over half of the analyzed samples were peripheral blood (52%), and the remaining samples were either bone marrow aspirate (46%) or from leukapheresis (2%). Most of the specimens were collected at initial acute leukemia diagnosis (62.2%), others were collected with residual disease or post chemotherapy (25.1%). The mean survival for this cohort was 13.7 months. Above median expression of Mcl1 was associated with longer survival (33 vs. 20 months; p = 0.0262). Above median expression of Bax was also associated with longer survival (32 vs. 18.2 months; p = 0.0114). In the Bax analysis, gene expression was further subdivided into 4 quartiles which showed correlation of expression levels with length of survival (Figure 1). The prognostic significance of the ratio Bax/ (Mcl1+Bcl2+Bcl-xL) was also analyzed given the mechanism of action of these various regulators. A higher ratio was associated with longer survival (40.7 vs. 18.7 months; p = 0.0095) (Figure 2) Conclusions Bcl-2, Bcl-xL and Mcl-1 are commonly expressed antiapoptotic proteins, whereas Bax is a promoter of apoptosis. The results with Bax are consistent with previous literature. Our results with Mcl1 expression differed from what we may have expected. This may be due to the sample size or methodology. However, it may also be due to the fact that a ratio of the actors is needed to help predict prognosis (as we analyzed in Figure 2). The overall survival between Bcl-2 and Bcl-xL was compared independently, however, they did not appear to have a significant difference in survival between high and low expression groups. We are currently working to see if this ratio can be validated in a prospective cohort of relapsed/ refractory AML patients treated on clinical trial and this data will be presented at the meeting. The Bcl-2 inhibitor venetoclax has demonstrated promising results in treating AML. However, venetoclax appears to have limited activity as a single agent. Our study suggests that potentially a combination of agents targeting factors such as Bcl-2, Bcl-xL and Mcl-1 may be associated with improved survival in patients with AML. Further validation of these biomarkers is warranted, and Mcl-1 inhibitors are currently in clinical trials.

A Novel Selective BCL2 Inhibitor with Limited Immune Suppression and Improved Safety Compared to Venetoclax

BCL2 is a key pro-survival protein that is overexpressed in many cancers. The first generation BCL2 inhibitor venetoclax has proved highly effective in multiple hematological malignancies including CLL and AML. However, the pharmacokinetic half-life of venetoclax is 26 hours which prevents optimization of the therapeutic window. Additionally, venetoclax metabolism is influenced by Cyp3A/4 inhibitors making their use in diseases where anti-fungal prophylaxis is required challenging. Venetoclax treatment can also result in significant immunosuppression including dose-limiting neutropenia and thrombocytopenia, and the loss of multiple lymphocyte populations, due in part to its effect on BCL-xL. Herein we present the pre-clinical evaluation of a novel highly selective BCL2 inhibitor, ZE50-0134, with equivalent anti-tumor efficacy, improved safety profile, and limited impact on non-malignant immune populations. ZE50-0134 binds the P2 pocket of BCL2, thereby increasing selectivity toward BCL-2, ZE50-0134 showed a 4600-fold greater selectivity for BCL2 over BCL-xL compared to venetoclax with only an 84-fold greater selectivity. In vivo murine and canine pharmacokinetics showed that ZE50-0134 has a substantially shorter half-life than venetoclax suggesting feasibility for periodic pulse dosing to limit the on-target adverse effects of BCL2 inhibition. The ADME properties of ZE50-0134 were exceptional. An in vivo murine study examining the pharmacology of ZE50-0134 or venetoclax given with ketoconazole, a strong CYP3A inhibitor, was performed demonstrating a 0.84-fold decrease in AUC with ZE50-0134 vs. a 1.52-fold increase in AUC with venetoclax suggesting less Cyp3A influence on ZE50-0134. Toxicology in rats and dogs suggest a 19-fold margin between therapeutically effective dose and toxicity with ZE50-0134. Given this data, we sought to validate that ZE50-0134 has less impact on normal immune populations. Wild-type C57BL6/J mice were treated daily with 100 mg/kg ZE50-0134, 100 mg/kg venetoclax or vehicle. A comprehensive 36-color spectral flow cytometry immune profiling panel was used to evaluate the effect of BCL2 inhibitors on multiple immune populations including B, T and NK cells as well as monocyte/macrophage and dendritic cell subsets in the peripheral blood. Venetoclax treated mice demonstrated a significant loss of multiple lymphocyte populations, including B, T, and NK cells, in the peripheral blood. Particularly, from Day 14 venetoclax treatment significantly reduced the percentage and absolute count of CD8 T cells (Figure 1). Some reduction in CD8 T cells was also observed with ZE50-0134 treatment, however it was significantly less than with venetoclax. No changes in the frequency of any normal myeloid population were observed. We next sought to examine the effectiveness of ZE50-0134 in B-cell and myeloid malignancies. Primary CLL cells were treated for 4 and 24 hours, corresponding to the expected half-life of ZE50-0134 and venetoclax, followed by washout. Cytotoxicity at 72 hours for both ZE50-0134 and venetoclax were equivalent for both short and prolonged treatment. We then assessed ZE50-0134 and venetoclax in vivo at 100 mg/kg in the RSV4;11 subcutaneous lymphoid model where we observed similar tumor growth regression. In AML, venetoclax is typically not effective as monotherapy and if often used in combination with azacytidine. To assess the relative efficacy of venetoclax and ZE50-0134 in combination with Azacitidine (5-Aza), we used the MOLM-13 cell line disseminated xenograft model. NCG mice were engrafted with MOLM-13 cells via the tail vein. From day 3, mice were treated with daily oral gavage of venetoclax or ZE50-0134 (100 mg/kg) alone or in combination with weekly intravenous 5-Aza (2.5 mg/kg). ZE50-0134 and venetoclax performed equally as single agents (median survival 23.5 days vs 22.5 days) and in combination with 5-Aza (median survival 35 days vs 35 days) (Figure 2). Together, this data suggests that ZE50-0134 is a highly effective BCL2 inhibitor with substantially less impact on the normal immune cells responsible for both anti-tumor and anti-infectious immunity. The pharmacology of this agent makes it an ideal therapeutic to optimize tumor BCL2 dependence and avoid untoward toxicity that occurs through continuous BCL2 targeting. This data supports the further clinical development of ZE50-0134 in both B cell malignancies and AML.

A Novel Tri-Hydroxy-Methylated Chalcone Isolated from Chromolaena tacotana with Anti-Cancer Potential Targeting Pro-Survival Proteins.

Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana) contains bioactive flavonoids that may have antioxidant and/or anti-cancer properties. This study investigated the potential anti-cancer properties of a newly identified chalcone isolated from the inflorescences of the plant Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana). The chalcone structure was determined using HPLC/MS (QTOF), UV, and NMR spectroscopy. The compound cytotoxicity and selectivity were evaluated on prostate, cervical, and breast cancer cell lines using the MTT assay. Apoptosis and autophagy induction were assessed through flow cytometry by detecting annexin V/7-AAD, active Casp3/7, and LC3B proteins. These results were supported by Western blot analysis. Mitochondrial effects on membrane potential, as well as levels of pro- and anti-apoptotic proteins were analyzed using flow cytometry, fluorescent microscopy, and Western blot analysis specifically on a triple-negative breast cancer (TNBC) cell line. Furthermore, molecular docking (MD) and molecular dynamics (MD) simulations were performed to evaluate the interaction between the compounds and pro-survival proteins. The compound identified as 2',3,4-trihydroxy-4',6'-dimethoxy chalcone inhibited the cancer cell line proliferation and induced apoptosis and autophagy. MDA-MB-231, a TNBC cell line, exhibited the highest sensitivity to the compound with good selectivity. This activity was associated with the regulation of mitochondrial membrane potential, activation of the pro-apoptotic proteins, and reduction of anti-apoptotic proteins, thereby triggering the intrinsic apoptotic pathway. The chalcone consistently interacted with anti-apoptotic proteins, particularly the Bcl-2 protein, throughout the simulation period. However, there was a noticeable conformational shift observed with the negative autophagy regulator mTOR protein. Future studies should focus on the molecular mechanisms underlying the anti-cancer potential of the new chalcone and other flavonoids from Ch. tacotana, particularly against predominant cancer cell types.

Molecular properties prediction, anticancer and anti-inflammatory activities of some pyrimido[1,2-b]pyridazin-2-one derivatives.

The anticancer and anti-inflammatory activities of a novel series of eleven pyrimido[1,2-b]pyridazin-2-one analogues substituted at position 7 were assessed in the current study. The physicochemical characteristics were studied using MolSoft software. The antiproliferative activity was investigated by MTT cell viability assay, and cell cycle analysis elucidated the antiproliferative mechanism of action. Western blot analysis examined the expression levels of key pro-apoptotic (Bax, p53) and pro-survival (Bcl-2) proteins. The anti-inflammatory activity was assessed by measuring the production levels of nitric oxide in RAW264.7 cells, and the expression levels of COX-2 enzyme in LPS-activated THP-1 cells. In addition, the gene expression of various pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α) and chemokines (CCL2, CXCL1, CXCL2, CXCL3) was assessed by RT-qPCR. Compound 1 bearing a chlorine substituent displayed the highest cytotoxic activity against HCT-116 and MCF-7 cancer cells where IC50 values of 49.35 ± 2.685 and 69.32 ± 3.186 µM, respectively, were achieved. Compound 1 increased the expression of pro-apoptotic proteins p53 and Bax while reducing the expression of pro-survival protein Bcl-2. Cell cycle analysis revealed that compound 1 arrested cell cycle at the G0/G1 phase. Anti-inflammatory assessments revealed that compound 1 displayed the strongest inhibitory activity on NO production with IC50 of 29.94 ± 2.24 µM, and down-regulated the expression of COX-2. Compound 1 also induced a statistically significant decrease in the gene expression of various cytokines and chemokines. These findings showed that the pyrimidine derivative 1 displayed potent anti-inflammatory and anticancer properties in vitro, and can be selected as a lead compound for further investigation.

USP51 facilitates colorectal cancer stemness and chemoresistance by forming a positive feed-forward loop with HIF1A.

In the current study, we have shown that USP51 promotes colorectal cancer stemness and chemoresistance, and high expression of USP51 predicts survival disadvantage in colorectal cancer patients. Mechanically, USP51 directly binds to Elongin C (ELOC) and forms a larger functional complex with VHL E3 ligase (USP51/VHL/CUL2/ELOB/ELOC/RBX1) to regulate the ubiquitin-dependent proteasomal degradation of HIF1A. USP51 efficiently deubiquitinates HIF1A and activates hypoxia-induced gene transcription. Conversely, the activation of HIF1A under hypoxia transcriptionally upregulates the expression of USP51. Thus, USP51 and HIF1A form a positive feedback loop. Further, we found that the SUMOylation of ELOC at K32 inhibits its binding to USP51. SUMO-specific protease 1 (SENP1) mediates the deSUMOylation of ELOC, promoting the binding of USP51 to ELOC and facilitating the deubiquitination and stabilization of HIF1A by USP51. Importantly, USP51 plays a crucial role in promoting the HIF1A and SENP1-dependent proliferation, migration, stemness, and chemoresistance under hypoxia in colorectal cancer. Together, our data revealed that USP51 is an oncogene stabilizing the pro-survival protein HIF1A, offering a potential therapeutic target for colorectal cancer.

Pro-Apoptotic Activity of MCL-1 Inhibitor in Trametinib-Resistant Melanoma Cells Depends on Their Phenotypes and Is Modulated by Reversible Alterations Induced by Trametinib Withdrawal.

Although BRAFV600/MEK inhibitors improved the treatment of melanoma patients, resistance is acquired almost inevitably. Trametinib withdrawal/rechallenge and MCL-1 inhibition in trametinib-resistance models displaying distinct p-ERK1/2 levels were investigated. Trametinib withdrawal/rechallenge caused reversible changes in ERK1/2 activity impacting the balance between pro-survival and pro-apoptotic proteins. Reversible alterations were found in MCL-1 levels and MCL-1 inhibitors, BIM and NOXA. Taking advantage of melanoma cell dependency on MCL-1 for survival, we used S63845. While it was designed to inhibit MCL-1 activity, we showed that it also significantly reduced NOXA levels. S63845-induced apoptosis was detected as the enhancement of Annexin V-positivity, caspase-3/7 activation and histone H2AX phosphorylation. Percentages of Annexin V-positive cells were increased most efficiently in trametinib-resistant melanoma cells displaying the p-ERK1/2low/MCL-1low/BIMhigh/NOXAlow phenotype with EC50 values at concentrations as low as 0.1 μM. Higher ERK1/2 activity associated with increased MCL-1 level and reduced BIM level limited pro-apoptotic activity of S63845 further influenced by a NOXA level. Our study supports the notion that the efficiency of an agent designed to target a single protein can largely depend on the phenotype of cancer cells. Thus, it is important to define appropriate phenotype determinants to stratify the patients for the novel therapy.

Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4+ Tcells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4+ Tcells from PLWH exvivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.

Anti-angiogenic therapy using the multi-tyrosine kinase inhibitor Regorafenib enhances tumor progression in a transgenic mouse model of ß-cell carcinogenesis

BackgroundPancreatic neuroendocrine tumors (PNETs) represent a distinct hypervascularized tumor entity, often diagnosed at metastatic stage. Therapeutic efficacy of anti-angiogenic multi-kinase inhibitors is frequently limited by primary or acquired resistance in vivo. This study aimed to characterize the molecular mode of action as well as resistance mechanisms to the anti-angiogenic multi-tyrosine kinase inhibitor (TKI) Regorafenib in vitro and in vivo.MethodsIn vitro, human and murine pancreatic neuroendocrine cell lines were comparatively treated with Regorafenib and other TKIs clinically used in PNETs. Effects on cell viability and proliferation were analyzed. In vivo, transgenic RIP1Tag2 mice were treated with Regorafenib at two different time periods during carcinogenesis and its impact on angiogenesis and tumor progression was evaluated.ResultsCompared to the established TKI therapies with Sunitinib and Everolimus, Regorafenib showed the strongest effects on cell viability and proliferation in vitro, but was unable to induce apoptosis. Unexpectedly and in contrast to these in vitro findings, Regorafenib enhanced proliferation during early tumor development in RIP1Tag2 mice and had no significant effect in late tumor progression. In addition, invasiveness was increased at both time points. Mechanistically, we could identify an upregulation of the pro-survival protein Bcl-2, the induction of the COX2-PGE2-pathway as well as the infiltration of CSF1R positive immune cells into the tumors as potential resistance mechanisms following Regorafenib treatment.DiscussionOur data identify important tumor cell-autonomous and stroma-dependent mechanisms of resistance to antiangiogenic therapies.

Inhibition of SIRT2 promotes death of human cytomegalovirus-infected peripheral blood monocytes via apoptosis and necroptosis

Peripheral blood monocytes are the cells predominantly responsible for systemic dissemination of human cytomegalovirus (HCMV) and a significant cause of morbidity and mortality in immunocompromised patients. HCMV establishes a silent/quiescent infection in monocytes, which is defined by the lack of viral replication and lytic gene expression. The absence of replication shields the virus within infected monocytes from the current available antiviral drugs that are designed to suppress active replication. Our previous work has shown that HCMV stimulates a noncanonical phosphorylation of Akt and the subsequent upregulation of a distinct subset of prosurvival proteins in normally short-lived monocytes. In this study, we found that SIRT2 activity is required for the unique activation profile of Akt induced within HCMV-infected monocytes. Importantly, both therapeutic and prophylactic treatment with a novel SIRT2 inhibitor, FLS-379, promoted death of infected monocytes via both the apoptotic and necroptotic cell death pathways. Mechanistically, SIRT2 inhibition reduced expression of Mcl-1, an Akt-dependent antiapoptotic Bcl-2 family member, and enhanced activation of MLKL, the executioner kinase of necroptosis. We have previously reported HCMV to block necroptosis by stimulating cellular autophagy. Here, we additionally demonstrate that inhibition of SIRT2 suppressed Akt-dependent HCMV-induced autophagy leading to necroptosis of infected monocytes. Overall, our data show that SIRT2 inhibition can simultaneously promote death of quiescently infected monocytes by two distinct death pathways, apoptosis and necroptosis, which may be vital for limiting viral dissemination to peripheral organs in immunosuppressed patients.

CDK9 INHIBITORS: a promising combination partner in the treatment of hematological malignancies.

Most hematological malignancies are characterized by overexpression of certain cancer promoting genes, such as MYC, MCL1 and cyclin D1. Preclinical studies in animal models have shown that CDK9 inhibitors supress the transcription of these anti-apoptotic and pro-survival proteins, and suggest their potential synergism with other drugs. In its first in-human trial, enitociclib demonstrated clinical activity in a small cohort of patients with high grade B lymphoma with MYC and BCL2 and/or BCL6 rearrangements, inducing complete responses in 2 of 7 subjects (29%) in monotherapy. These data suggest CDK9 inhibitors could play a role in the treatment of hematological diseases and could be a great ally when combined with other therapeutic approaches.

Protection of high-frequency low-intensity pulsed electric fields and brain-derived neurotrophic factor for SH-SY5Y cells against hydrogen peroxide-induced cell damage.

Neurodegenerative diseases (NDDs) pose a significant global health threat. In particular, Alzheimer disease, the most common type causing dementia, remains an incurable disease. Alzheimer disease is thought to be associated with an imbalance of reactive oxygen species (ROS) in neurons, and scientists considered ROS modulation as a promising strategy for novel remedies. In the study, human neural cell line SH-SY5Y was used in probing the effect of combining noninvasive high-frequency low-intensity pulsed electric field (H-LIPEF) and brain-derived neurotrophic factor (BDNF) in protection against hydrogen peroxide (H2O2)-induced neuron damage. Our result finds that the combination approach has intensified the neuroprotective effect significantly, perhaps due to H-LIPEF and BDNF synergistically increasing the expression level of the phosphorylated epidermal growth factor receptor (p-EGFR), which induces the survival-related mitogen-activated protein kinases (MAPK) proteins. The study confirmed the activation of extracellular signal-regulated kinase (ERK) and the downstream pro-survival and antioxidant proteins as the mechanism underlying neuron protection. These findings highlighted the potential of H-LIPEF combined with BDNF in the treatment of NDDs. Furthermore, BDNF-mimetic drugs combining with noninvasive H-LIPEF to patients is a promising approach worthy of further research. This points to strategies for selecting drugs to cooperate with electric fields in treating neurodegenerative disorders.

Bcl-2 supports survival and metabolic fitness of quiescent tissue-resident ILC3

Group 3 innate lymphoid cells (ILC3) are potent effector cells with critical roles in enforcing immunity, barrier integrity and tissue homeostasis along the gastrointestinal tract. ILC3 are considered primarily tissue-resident cells, seeding the gastrointestinal tract during embryonic stages and early life. However, the mechanisms through which mature ILC3 are maintained within adult tissues are poorly understood. Here, we report that lymphoid tissue-inducer-like (LTi-like) ILC3 exhibit minimal turnover in the healthy adult intestinal tract, persist for extended periods of time, and display a quiescent phenotype. Strikingly, during enteric bacterial infection LTi-like ILC3 also exhibit negligible hematopoietic replenishment and remain non-proliferative, despite robustly producing cytokines. Survival of LTi-like ILC3 was found to be dependent upon the balance between the metabolic activity required to drive effector function and anti-apoptotic programs. Notably, the pro-survival protein B-cell lymphoma-2 (Bcl-2) was required for the survival of LTi-like ILC3 ex vivo but was rendered partially dispensable if mitochondrial respiration was inhibited. Together we demonstrate LTi-like ILC3 are a tissue-resident, quiescent population that persist independently of hematopoietic replenishment to survive within the intestinal microenvironment.

Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment.

Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1β cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.

Hippocampal delivery of neurotrophic factor-α1/carboxypeptidase E gene prevents neurodegeneration, amyloidosis, memory loss in Alzheimer’s Disease male mice

Alzheimer’s Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aβ1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aβ1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aβ1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation.

Targeting Exportin 1 as treatment for cutaneous squamous cell carcinoma.

e21564 Background: Cutaneous squamous cell carcinomas (cSCC) are among the most frequent solid cancers in humans and in the U.S., cSCC cases are increasing. Molecular mechanisms transitioning premalignant lesions to cancer are not well characterized. As a result, targeted therapies need to be identified to treat these lesions. Preliminary data shows that Exportin 1 (XPO1) is one of the major proteins regulating nuclear export of proteins, helping to maintain cellular homeostasis. Previous work showed that Exportin 1 modulates the nuclear:cytoplasmic ratio of signaling proteins in skin cancer; resulting in an anti-apoptotic effect by adjusting the nuclear-cytoplasmic balance of pro-survival proteins such as CDC25A and Survivin. XPO1 expression is upregulated in many cancers, thereby affecting the nuclear:cytoplasmic ratio of many pro-survival proteins and enhancing tumorigenesis. This research project was designed to investigate the effects of XPO1 inhibition on cSCC survival. Selinexor is FDA cleared for treatment of multiple myeloma patients. Selinexor is a selective inhibitor of XPO1 regulated nuclear transport causing target molecules to remain within the cell leading to decreased cancer cell growth and decreased cancer cell survival. We hypothesize that inhibiting XPO1 with Selinexor or by CRISPRi genetic knockdown of XPO1 in cSCC cells will reduce proliferation and increase apoptosis in cSCC cells. Methods: Human cSCC cell lines SCC13 and SRB12 were seeded in 96-well plates at 8,000 cells/well density and 3,000 cells/well, respectively. Cells were treated with varying concentrations of Selinexor (0.005mM – 5mM) or DMSO as vehicle control. At 24-hour intervals, cell imaging and Neutral red staining assays were conducted to determine the IC50 concentration for Selinexor in cSCC cell lines. SCC13 cells stably expressing dCas9-KRAB and a doxycycline-inducible XPO1 guide RNA were used for CRISPRi experiments. Cells were treated with doxycycline daily and apoptosis was assayed by Caspase Glo 3/7 daily for four days. CRIPSRi enabled a doxycycline inducible XPO1 knock down on SCC13 cells allowing us to disrupt the cellular transporter protein XPO1. Results: Selinexor treatment of SRB12 cells resulted in an IC50 of 1.174 μM and 0.071 μM for SCC13. In contrast, normal human keratinocyte cell lines had higher IC50 values of 2.18 μM for HaCaT and 5.29 μM for KerCT cells. Apoptosis significantly increased after 72 and 96 hours of XPO1 knock down in SCC13 cells measured by Caspase Glo. Conclusions: These results document how XPO1 inhibition reduces proliferation and is toxic to both normal keratinocytes and cSCC cells. Additionally, cSCC cells had lower IC50 values for Selinexor, suggesting cSCC may be more sensitive to XPO1 inhibition compared to normal keratinocytes. Further, targeting XPO1 also increases apoptosis in these cells. This data supports the hypothesis that targeting XPO1 may be effective in treating cSCC.

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

A bi-steric mTORC1-selective inhibitor overcomes drug resistance in breast cancer

Activation of the PI3K-mTOR pathway is central to breast cancer pathogenesis including resistance to many targeted therapies. The mTOR kinase forms two distinct complexes, mTORC1 and mTORC2, and understanding which is required for the survival of malignant cells has been limited by tools to selectively and completely impair either subcomplex. To address this, we used RMC-6272, a bi-steric molecule with a rapamycin-like moiety linked to an mTOR active-site inhibitor that displays >25-fold selectivity for mTORC1 over mTORC2 substrates. Complete suppression of mTORC1 by RMC-6272 causes apoptosis in ER+/HER2− breast cancer cell lines, particularly in those that harbor mutations in PIK3CA or PTEN, due to inhibition of the rapamycin resistant, mTORC1 substrate 4EBP1 and reduction of the pro-survival protein MCL1. RMC-6272 reduced translation of ribosomal mRNAs, MYC target genes, and components of the CDK4/6 pathway, suggesting enhanced impairment of oncogenic pathways compared to the partial mTORC1 inhibitor everolimus. RMC-6272 maintained efficacy in hormone therapy-resistant acquired cell lines and patient-derived xenografts (PDX), showed increased efficacy in CDK4/6 inhibitor treated acquired resistant cell lines versus their parental counterparts, and was efficacious in a PDX from a patient experiencing resistance to CDK4/6 inhibition. Bi-steric mTORC1-selective inhibition may be effective in overcoming multiple forms of therapy-resistance in ER+ breast cancers.

Therapeutic inhibition of Bmi-1 ablates chemoresistant cancer stem cells in adenoid cystic carcinoma

ObjectivesAdenoid Cystic Carcinomas (ACC) typically show modest responseto cytotoxic therapy. Cancer stem cells (CSC) have been implicated in chemoresistance and tumor relapse. However, their role in ACC remains unknown. The purpose of this work was to evaluate the impact of targeting ACC CSCs with Bmi-1 inhibitors on resistance to cytotoxic therapy and tumor relapse. Materials and methodsTherapeutic efficacy of a small molecule inhibitor of Bmi-1 (PTC596; Unesbulin) and/or Cisplatin on ACC stemness was evaluated in immunodeficient mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in human ACC cell-lines (UM-HACC-2A,-14) or low passage primary human ACC cells (UM-HACC-6). The effect of therapy on stemness was examined by salisphere assays, flow cytometry for ALDH activity and CD44 expression, and Western blots for Bmi-1 (self-renewal marker) and Oct4 (embryonic stem cell marker) expression. ResultsPlatinum-based agents (Cisplatin, Carboplatin) induced Bmi-1 and Oct4 expression, increased salisphere formation and the CSC fraction in vitro and in vivo. In contrast, PTC596 inhibited expression of Bmi-1, Oct4 and pro-survival proteins Mcl-1 and Claspin; decreased the number of salispheres, and the fraction of ACC CSCs in vitro. Silencing Claspin decreased salisphere formation and CSC fraction. Both, single agent PTC596 and PTC596/Cisplatin combination decreased the CSC fraction in PDX ACC tumors. Notably, short-term combination therapy (2 weeks) with PTC596/Cisplatin prevented tumor relapse for 150 days in a preclinical trial in mice. ConclusionTherapeutic inhibition of Bmi-1 ablates chemoresistant CSCs and prevents ACC tumor relapse. Collectively, these results suggest that ACC patients might benefit from Bmi-1-targeted therapies.