Mcl-1 Reduction Research Articles

BRD4 Levels Determine the Response of Human Lung Cancer Cells to BET Degraders That Potently Induce Apoptosis through Suppression of Mcl-1.

Lung cancer consists of approximately 80% non-small cell lung cancer (NSCLC) and 20% small cell lung cancer (SCLC) and remains the leading cause of cancer-related deaths worldwide despite advances in early diagnosis, targeted therapy, and immunotherapy. Thus, novel therapies are still urgently needed. Bromodomain and extraterminal (BET) proteins, primarily comprised of BRD2, BRD3, and BRD4 proteins, function as epigenetic readers and master transcription coactivators and are now recognized cancer therapeutic targets. BET degraders such as ZBC260 and dBET represent a novel class of BET inhibitors that act by inducing BET degradation. The current study demonstrates the therapeutic efficacies of BET degraders, particularly ZBC260, against lung cancer, as well as understanding the underlying mechanisms and identifying molecular markers that determine cell sensitivity to BET degraders. A panel of NSCLC cell lines possessed similar response patterns to ZBC260 and dBET but different responses to BET inhibitor JQ-1. BRD levels, particularly BRD4, correlated positively with high sensitivity to BET degraders but not to JQ-1. BET degraders potently induced apoptosis in sensitive NSCLC cells and were accompanied by reduction of Mcl-1 and c-FLIP levels, which are critical for mediating induction of apoptosis and enhancement of TRAIL-induced apoptosis. Accordingly, ZBC260 exerted more potent activity than JQ-1 in vivo against the growth of NSCLC xenografts and patient-derived xenografts. These findings warrant future clinical validation of the efficacy of BET degraders in NSCLC, particularly those with high levels of BRD proteins, especially BRD4. SIGNIFICANCE: The current study demonstrates the potential of novel BET degraders in the treatment of lung cancer and warrants clinical validation of BET degraders in lung cancer with high levels of BRD4.

Inhibition of EGFR Signaling and Activation of Mitochondrial Apoptosis Contribute to Tanshinone IIA-Mediated Tumor Suppression in Non-Small Cell Lung Cancer Cells.

BackgroundDeregulation of epidermal growth factor receptor (EGFR) signaling plays a critical role in non-small cell lung cancer (NSCLC) tumorigenesis. The natural product Tanshinone IIA (Tan IIA) exhibits significant anti-tumor effect in various human cancers, however, the mechanism remains elusive.MethodsThe inhibitory effect of Tan IIA NSCLC cells was determined by MTS and soft agar assays. The activation of EGFR signaling and the protein level of myeloid cell leukemia 1 (Mcl-1) were examined by immunoblot (IB), immunohistochemical staining (IHC), and ubiquitination analysis. The in vivo anti-tumor effect was validated by the xenograft mouse model.ResultsTan IIA inhibits NSCLC cells through suppression of EGFR signaling. Tan IIA decreases cell viability and colony formation in EGFR wild type and activating mutant cell lines. The IB data further confirmed that Tan IIA suppresses EGFR phosphorylation time- and dose-dependently. Tan IIA destabilizes Mcl-1 and shortens the half-life. Ubiquitination analysis showed that treatment with Tan IIA promotes Mcl-1 ubiquitination and degradation. Further study showed that the downregulation of EGFR-Akt signaling is required for Tan IIA-induced Mcl-1 reduction. Ectopic overexpression of constitutively activated Akt1 compromised these antitumor efficacies in Tan IIA-treated NSCLC cells. Finally, Tan IIA inhibited the in vivo tumor growth.ConclusionOur data indicate that Tan IIA acts as an EGFR signaling inhibitor, and targeting EGFR-Akt-Mcl1 axis could provide a new option for NSCLC treatment.

Overcoming acquired resistance of EGFR-mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib, with the natural product honokiol.

The development of acquired resistance to osimertinib (Osim) (AZD9291 or TAGRISSOTM), an FDA‐approved third‐generation epidermal growth factor receptor (EGFR) inhibitor for the treatment of EGFR‐mutant nonsmall cell lung cancer (NSCLC), limits the long‐term benefits for patients. Thus, effective treatment options are urgently needed. To this end, we explored whether honokiol (HNK), a natural product with potential antitumor activity, may be used to overcome Osim resistance. The combination of HNK and Osim synergistically decreased the survival of several Osim ‐resistant cell lines with enhanced effects on inhibiting cell colony formation and growth and on inducing apoptosis. This combination also showed greater growth suppression of Osim‐resistant xenograft tumors including those with 19del, T790M, and C797S triple mutations in nude mice. Mechanistically, the augmented induction of apoptosis by the combination is largely due to enhanced Mcl‐1 reduction through facilitating its degradation. A synthetic HNK derivative exerted similar effects with greater efficacy. Our findings warrant further study of HNK and its derivatives in overcoming Osim resistance in the clinic.

Rational Combination Therapy for Melanoma with Dinaciclib by Targeting BAK-Dependent Cell Death.

Mutation of the oncogene BRAF is among the most common genetic alterations in melanoma. BRAF inhibitors alone or in combination with MEK inhibitors fail to eradicate the tumor in most patients due to combinations of intrinsic or acquired resistance. Therefore, novel strategies are needed to improve the therapeutic efficacy of BRAF inhibition. We demonstrated that dinaciclib has potent antimelanoma effects by inducing BAK-dependent apoptosis through MCL1 reduction. Contrary to dinaciclib, the inhibitors of BRAF/MEK/CDK4/6 induced apoptosis dominantly through a BAX-dependent mechanism. Although the combination of BRAF and MEK inhibitors did not exhibit additive antimelanoma effects, their combination with dinaciclib synergistically inhibited melanoma growth both in vitro and in vivo Collectively, our present findings suggest dinaciclib to be an effective complementary drug of BAX-dependent antimelanoma drugs by targeting BAK-mediated apoptosis, and other such rational drug combinations can be determined by identifying complementary drugs activating either BAK or BAX.

Deguelin suppresses non-small cell lung cancer by inhibiting EGFR signaling and promoting GSK3\u03b2/FBW7-mediated Mcl-1 destabilization

Activating mutations of epidermal growth factor receptor (EGFR) play crucial roles in the oncogenesis of human non-small cell lung cancer (NSCLC). By screening 79 commercially available natural products, we found that the natural compound deguelin exhibited a profound anti-tumor effect on NSCLC via directly down-regulating of EGFR-signaling pathway. Deguelin potently inhibited in vitro EGFR kinase activity of wild type (WT), exon 19 deletion, and L858R/T790M-mutated EGFR. The in silico docking study indicated that deguelin was docked into the ATP-binding pocket of EGFRs. By suppression of EGFR signaling, deguelin inhibited anchorage-dependent, and independent growth of NSCLC cell lines, and significantly delayed tumorigenesis in vivo. Further study showed that deguelin inhibited EGFR and downstream kinase Akt, which resulted in the activation of GSK3β and eventually enhanced Mcl-1 phosphorylation at S159. Moreover, deguelin promoted the interaction between Mcl-1 and E3 ligase SCFFBW7, which enhanced FBW7-mediated Mcl-1 ubiquitination and degradation. Additionally, phosphorylation of Mcl-1 by GSK3β is a prerequisite for FBW7-mediated Mcl-1 destruction. Depletion or pharmacological inactivation of GSK3β compromised deguelin-induced Mcl-1 ubiquitination and reduction. Taken together, our data indicate that enhancement of ubiquitination-dependent Mcl-1 turnover might be a promising approach for cancer treatment.

The Anti-Proliferative Activity of the Hybrid TMS-TMF-4f Compound Against Human Cervical Cancer Involves Apoptosis Mediated by STAT3 Inactivation.

We previously reported the potential anti-proliferative activity of 3-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-N-(3,4,5-trimethoxyphenyl) benzamide (TMS-TMF-4f) against human cancer cells; however, the underlying molecular mechanisms have not been investigated. In the present study, TMS-TMF-4f showed the highest cytotoxicity in human cervical cancer cells (HeLa and CaSki) and low cytotoxicity in normal ovarian epithelial cells. Annexin V-FITC and propidium iodide (PI) double staining revealed that TMS-TMF-4f-induced cytotoxicity was caused by the induction of apoptosis in both HeLa and CaSki cervical cancer cells. The compound TMS-TMF-4f enhanced the activation of caspase-3, caspase-8, and caspase-9 and regulated Bcl-2 family proteins, which led to mitochondrial membrane potential (MMP) loss and resulted in the release of cytochrome c and Smac/DIABLO into the cytosol. Also, TMS-TMF-4f suppressed both constitutive and IL-6-inducible levels of phosphorylated STAT3 (p-STAT3) and associated proteins such as Mcl-1, cyclin D1, survivin, and c-Myc in both cervical cancer cells. STAT-3 overexpression completely ameliorated TMS-TMF-4f-induced apoptotic cell death and PARP cleavage. Docking analysis revealed that TMS-TMF-4f could bind to unphosphorylated STAT3 and inhibit its interconversion to the activated form. Notably, intraperitoneal administration of TMS-TMF-4f (5, 10, or 20 mg/kg) decreased tumor growth in a xenograft cervical cancer mouse model, demonstrated by the increase in TUNEL staining and PARP cleavage and the reduction in p-STAT3, Mcl-1, cyclin D1, survivin, and c-Myc expression levels in tumor tissues. Taken together, our results suggest that TMS-TMF-4f may potentially inhibit human cervical tumor growth through the induction of apoptosis via STAT3 suppression.

Abstract 1277: Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

Abstract Osimertinib (AZD9291) is a FDA-approved EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line). The development of acquired resistance to osimertinib limits the long-term benefits of patients, and effective treatment options are urgently needed. We have identified that honokiol, a natural product with potential antitumor activity, may be used to overcome osimertinib resistance. In several osimertinib-resistant cell lines including HCC827/AR, PC-9/AR, PC-9/GR/AR and PC-9/3M (with EGFR 19del, T790M and C797S mutations), the combination of honokiol and osimertinib had synergistic effects on decreasing the survival of these cell lines. The combination inhibited cell colony formation and growth, and augmented induction of apoptosis of resistant cells. This combination enhanced reduction of Mcl-1, a critical mechanism by which osimertinib induces apoptosis in sensitive EGFR mutant NSCLC cells; this effect is likely due to enhanced Mcl-1 degradation since Mcl-1 was degraded much faster in cells exposed to the combination of honokiol and osimertinib than in DMSO-treated control cells. Moreover enforced overexpression of ectopic Mcl-1 abolished the enhanced effect of honokiol and osimertinib combination on induction of apoptosis, supporting a critical role of Mcl-1 reduction in inducing apoptosis of the osimertinib-resistant cells by this combination. Our findings warrant further study of this combination for overcoming osimertinib-resistance in vivo and potentially in the clinic. (This work was supported by the NIH/NCI R01 CA223220 to SYS and Winship lung cancer pilot award to SYS). Citation Format: Hongjing ZANG, Songqing Fan, Jack Arbiser, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Honokiol, a natural product, overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1277.

Abstract 77: Therapeutic potential of ERK inhibitors in overcoming acquired resistance to third generation EGFR tyrosine kinase inhibitors

Abstract Targeting epidermal growth factor receptor (EGFR) activating mutations, 90% of which present as an exon 19 deletion (Del19) or exon 21 point mutation (L858R), with first and second generation EGFR tyrosine kinase inhibitors (EGFR-TKIs; e.g., erlotinib and afatinib) and T790M resistant mutation with third generation EGFR-TKIs (e.g., AZD9291 or osimertinib) has provided significant clinical benefit in patients with non-small cell lung cancer (NSCLC) carrying these mutations. AZD9291 selectively and irreversibly inhibits EGFR activating and T790M mutants while sparing wild-type EGFR. It is now an approved therapeutic option for NSCLC patients with activating EGFR mutations (first-line) or those who have become resistant to the 1st generation EGFR-TKIs through the T790M mutation (second-line). Unfortunately, all patients eventually relapsed and developed resistance to AZD9291 treatment, limiting the long-term benefit of this targeted therapy. Hence the effective strategies that can overcome the resistance are urgently needed in the clinic. We have recently demonstrated that modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and loss of MEK/ERK-mediated Bim and Mcl-1 modulation is a key mechanism accounting for development of acquired resistance to AZD9291. Accordingly we have suggested that inhibition of the MEK/ERK signaling, which enforces Mcl-1 reduction and Bim elevation, might lower the threshold of AZD9291-resistant cells to re-respond to AZD9291 for undergoing apoptosis and achieve the goal of overcoming acquired resistance. Indeed, the combination of AZD9291 with a MEK inhibitor effectively decreases cell survival with enhanced apoptosis irrespectively of resistant mechanisms and inhibited the growth of AZD9291-resistant xenografts in vivo as we recently reported. In the current study, we further show that the combination of AZD9291 with an ERK inhibitor synergistically decreased the survival of AZD9291-resistance cell lines with enhanced induction of apoptosis. Moreover, the combination of a MEK or ERK inhibitor with a first (e.g., erlotinib) or second (e.g., afatinib) generation EGFR-TKIs also very effectively decreased the survival of AZD9291-resistance cell lines although these cell lines were cross-resistant to first and second generation EGFR-TKIs. Therefore, our results have provided additional evidence supporting the notion that co-targeting the MEK/ERK signaling is an effective strategy for overcoming AZD9291 acquired resistance irrespectively of the underlying resistance mechanisms. Our findings warrant further investigation of this therapeutic strategy to overcome AZD9291 resistance in the clinic. (This study was supported by the NIH/NCI R01 CA223220 and Winship lung cancer pilot award) Citation Format: Yiting Li, Guoqing Qian, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Therapeutic potential of ERK inhibitors in overcoming acquired resistance to third generation EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 77.

XPO1 inhibitor KPT-330 synergizes with Bcl-xL inhibitor to induce cancer cell apoptosis by perturbing rRNA processing and Mcl-1 protein synthesis

XPO1 (exportin1) mediates nuclear export of proteins and RNAs and is frequently overexpressed in cancers. In this study, we show that the orally bioavailable XPO1 inhibitor KPT-330 reduced Mcl-1 protein level, by which it synergized with Bcl-xL inhibitor A-1331852 to induce apoptosis in cancer cells. KPT-330/A-1331852 combination disrupted bindings of Mcl-1 and Bcl-xL to Bax, Bak, and/or Bim, elicited mitochondrial outer membrane permeabilization, and triggered apoptosis. KPT-330 generally mitigated mRNA expression and protein synthesis rather than mRNA nuclear export or protein stability of Mcl-1. KPT-330 inhibited mTORC1/4E-BP1 and Mnk1/eIF4E axes, which disrupted the eIF4F translation initiation complex but was dispensable for Mcl-1 reduction and KPT-330/A-1331852 combination-induced apoptosis. Mature rRNAs are integral components of the ribosome that determines protein synthesis ability. KPT-330 impeded nucleolar rRNA processing and reduced total levels of multiple mature rRNAs. Reconstitution of XPO1 by expressing degradation-resistant C528S mutant retained rRNA amount, Mcl-1 expression, and Bcl-xL inhibitor resistance upon KPT-330 treatment. KPT-330/A-1331852 combination suppressed growth and enhanced apoptosis of non-small cell lung cancer xenografts. Therefore, we clarify the reason of apoptosis resistance of cancer cells to XPO1 inhibition and develop a potential strategy for treating solid tumors.

Novel Synergistic Combination of Mitotic Arrest and Promotion of Apoptosis for Treatment of Pancreatic Adenocarcinoma

The BCL-2 family of proteins, including anti-apoptotic members BCL-2, BCL-XL and MCL-1, are part of a complex network that controls apoptosis. BH3-mimetics such as ABT-263 inhibit anti-apoptotic BCL-2 proteins and have been developed as potential cancer therapeutics. Aurora Kinase A (AKA) is over-expressed in pancreatic cancer (PC) and controls G2-M transition during mitosis and AKA inhibitors have been developed that induce mitotic arrest. We hypothesized that mitotic arrest induced by AKA inhibition may sensitize PC to accelerated apoptosis by a BH3-mimetic. Our results demonstrated that ABT-263 plus MLN8237 treatment showed greater activity than either single drug alone, as well as strong synergism, in the inhibition of growth of pancreatic cell lines (AsPC-1, PANC-1, MIA PaCa-2, HPAF-II) and PC patient-derived organoids (PDOs). The higher efficacy of combination treatment was attributable to the higher levels of induction of apoptosis and reduction of MCL-1 in PC cells and PDOs. In addition, combination therapy was more effective than single drug in the suppression of tumor growth in AsPC-1 xenograft mouse models. Together, our findings suggest that combination therapy with ABT-263 and MLN8237 should be considered for further exploration as a novel treatment of deadly PC disease.

Abstract 883: Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92

Abstract The positive-transcription elongation factor (P-TEFb) complex is critical in stimulating the transcription of non-abortive transcripts by RNA polymerase II. The CDK9/cyclin-T heterodimer comprises the P-TEFb complex and promotes transcription elongation through phosphorylation of serine 2 of the heptapeptide repeats of the c-terminal domain of RNA Polymerase II (RNAPII-CTD). Alvocidib, a potent CDK9 inhibitor, has been shown to suppress expression of MCL-1 and Myc mRNAs, and clinical pharmacodynamic data suggest that alvocidib's activity is primarily mediated through the suppression of MCL-1 mRNA and protein expression. In addition to MCL-1 suppression, we hypothesized that CDK9 inhibition affects other RNA Polymerase II target RNAs, such as microRNAs. Recently, it was reported that inhibition of bromodomain and extraterminal domain (BET) protein by JQ1 suppresses miR17-92 expression. miR17-92 negatively regulates expression of the pro-apoptotic BH3-only protein, BIM, leading to suppression of BIM expression, thereby decreasing the cells' ability to induce apoptosis. It was hypothesized that CDK9 kinase activity is key to miR17-92 expression and suppression of BIM expression and that targeting CDK9 would lead to a decrease in miR17-92, and increases in BIM. MV4-11, OCI-AML3, MOLM13, and THP1 AML cell lines were used to determine the effects of alvocidib treatment on microRNAs. RT-qPCR was utilized to determine microRNA expression levels of miR17-92 and mRNA levels of BIM and other markers. Protein changes were determined using standard gel electrophoresis and immunoblotting technique. CellTiter-Glo and Caspase-Glo were used for all cell viability and apoptosis assays interrogating alvocidib. With CDK9 inhibition mediated via alvocidib, a dose- and time-dependent decrease in miR17-92 expression in MV4-11, OCI-AML3, MOLM13, and THP1 cells was observed. A decrease was observed even 3 hours post-treatment, persisting for up to 24 hours. MicroRNA suppression following treatment ranged in magnitude, with a maximal effect of between 3.3 to 4.2-fold suppression, depending on the cell line and timepoint. This suppression coincided with an increase in BIM mRNA and protein expression in the MV4-11, OCI-AML3, and MOLM13 cells. However, BIM protein increase was not observed in THP1 cells. Maximal increase in BIM mRNA levels reached 5.4-fold in the MV4-11 cell line. Our data suggest that CDK9 inhibition suppresses RNA Polymerase II-mediated expression of miR17-92, which in turn leads to increased expression of BIM. Combined with MCL-1 reduction, increased BIM protein expression mediated by alvocidib leads to enhanced apoptosis. Taken together, the data provide additional understanding of CDK9 as a potential therapeutic target, and are consistent with the hypothesis that CDK9 activity is necessary for miR17-92 expression. Citation Format: Hillary Haws, Hubert F. Arokium, James L. Bogenberger, Adam Siddiqui-Jain, David J. Bearss, Raoul Tibes, Steven L. Warner, Clifford J. Whatcott. Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 883.

Abstract 297: AZD4573: Mechanistic PKPD model linking CDK9 inhibition to Mcl1 depletion and induction of apoptosis in preclinical AML model

Abstract Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition results in the selective downregulation of genes with short-lived transcripts and labile proteins - including the anti-apoptotic protein Mcl1. AZD4573 is a selective inhibitor of CDK9 with short pharmacokinetic (PK) half-life. Intermittent dosing of AZD4573 in mouse MV411 (AML cell line) xenograft models results in progressive reduction in tumor volume with the mechanism of action believed to be via induction of apoptosis following depletion of Mcl1. The aim of this work was to derive a quantitative understanding of the relationships between extent and duration of CDK9 inhibition, depletion of Mcl1 and rate of induction of apoptosis in MV411 tumor cells. A mechanistic model has been established that quantitatively and dynamically connects AZD4573 plasma and tumor PK to the rate and extent of modulation of pSer2-RNAPII and Mcl1 in the tumor and rate of induction of cell death (as measured by reduction in tumor volume). Tumor pSer2-RNAPII and Mcl1 pharmacodynamics were modeled using a series of linked indirect response models. Production rate of pSer2-RNAPII was modeled as being directly inhibited by AZD4573 concentration in the tumor. Production rate of Mcl1 was linked to pSer2-RNAPII via a series of transit compartments to capture the transcription/translation driven delay in onset of response. Induction of intrinsic apoptosis in the MV411 tumor cells was modelled as being inhibited by Mcl1. Tumor pSer2-RNAPII exhibited a rapid, dose-dependent decrease following IP dosing of AZD4573 in mice. The free concentration of AZD4573 that resulted in half-maximal inhibition of pSer2-RNAPII production rate was estimated to be in the range 11-21 nM. Following a brief delay, tumor Mcl1 also exhibited a relatively rapid decrease that was proportional to the pSer2-RNAPII response. Mcl1 protein half-life was estimated to be 0.3 hr. Rate of induction of apoptosis could be decribed as a saturable first-order process (Kmax ~ 0.2 hr-1) and appeared to exhibit a steep response to the depletion of Mcl1, with reduction of Mcl1 to 25% (of the baseline value) being estimated to result in half-maximal rate of induction of apoptosis in the MV411 cells. The described MV411 PKPD/efficacy model has been assumed to be representative of AML in human patients and was used to derive preliminary predictions of clinical efficacy at a range of possible IV dosing regimens. Citation Format: Douglas Ferguson, Theresa Proia, Justin Cidado, Scott Boiko, Maryann San Martin, Steven Criscione, Wenlin Shao, Lisa Drew. AZD4573: Mechanistic PKPD model linking CDK9 inhibition to Mcl1 depletion and induction of apoptosis in preclinical AML model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 297.

Novel therapeutic strategy to inhibit growth of pancreatic cancer organoids using a rational combination of drugs to induce mitotic arrest and apoptosis.

322 Background: The ability of cancer cells to suppress apoptosis is critical for carcinogenesis. The Bcl-2-family of regulator proteins, including the anti-apoptotic members Bcl-2, Bcl-xL and Mcl-1, contributes to a complex network in control of apoptosis. BH3-mimetics (e.g. ABT-263) can inhibit anti-apoptotic Bcl-2 proteins and therefore have been developed as potential cancer therapeutics. Aurora Kinase A (AKA) is over-expressed in pancreatic cancer (PC) and is expressed to regulate G2-M transition during mitosis, making it an attractive target for PC. In this study we hypothesized that a combination of mitotic arrest using an AKA inhibitor (e.g. MLN8237) would sensitize PC to induction of apoptosis by a BH3-mimetic. Methods: Pancreatic cell lines (AsPC-1, PANC-1, MIA PaCa-2, HPAF-II) and patient-derived pancreatic cancer organoids (PDO) were treated with a BH3-mimetic (ABT-263) alone, an AKA inhibitor (MLN8237) alone, or the combination in comparison to untreated controls. Cell viability was measured using the CellTiter-Fluor (Promega) assay. Apoptosis was evaluated by Western blot (WB) for cleaved PARP, caspase 3 or caspase 7, and flow cytometry. Nude mice were implanted with pancreatic cancer cells to generate PC xenografts which were then treated with the same 4 treatment groups as in the in vitro studies. Results: ABT-263 combined with MLN8237 showed greater potency than either single drug alone, demonstrating synergy in inhibiting the growth of PC cells and PDOs. Combined treatment with MLN8237 and ABT-263 in PDOs suppressed organoid formation and proliferation by inducing apoptosis. Mechanistically, MLN8237 enhanced the activity of ABT-263 through reduction of Bcl-xL and Mcl-1 in pancreatic cancer cell lines and PDOs. The combination therapy also showed greater suppression of the growth of xenograft tumors, as compared with control treatments with single drug alone or vehicle. Conclusions: The combination of ABT-263 and MLN8237 appears to synergistically induce apoptosis via reduction of Bcl-2 family proteins in PC and should be further explored.

Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation.

Purpose: The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study.Experimental Design: AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytometry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenograft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA.Results: AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivoConclusions: Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291. Clin Cancer Res; 23(21); 6567-79. ©2017 AACR.

Abstract A34: The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies

Abstract Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in >30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P<0.01). We also showed that the effect of ibrutinib on CD20 levels is mediated by inhibition of CXCR4/SDF-1 axis, which impairs the SDF-1α (ligand for CXCR4) mediated regulation of CD20 expression (P<0.01). Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after >3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P<0.01). This suggest that the observed in vivo up-regulation of specific Bcl2 family members is due to an adaptation of B cells or selection of malignant B cells with high-level expression of anti-apoptotic molecules sufficient to survive the BCR signalling inhibition. We observed that the survival of CLL cells treated with ibrutinib is dependent on Bcl2, and subsequent Bcl2 inhibition can act synergistically with ibrutinib in B cell lines and CLL samples. These anti-apoptotic molecules therefore represent primary candidates with synthetically lethal potential in combination with ibrutinib. Additionally, we have identified dependence of a subtype of CLL cases on integrin signalling. The inhibition of integrin VLA-4 with anti-VLA4 antibody or integrin-associated kinase overcame the resistance of CLL cells to chemotherapy drugs and also to anti-CD20 antibodies, and synergized with the effects of ibrutinib. Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

Dual targeting of MCL1 and NOXA as effective strategy for treatment of mantle cell lymphoma.

Imbalances in the composition of BCL2 family proteins contribute to tumourigenesis and therapy resistance of mantle cell lymphoma (MCL), making these proteins attractive therapy targets. We studied the efficiency of dual targeting the NOXA/MCL1 axis by combining fatty acid synthase inhibitors (NOXA stabilization) with the CDK inhibitor Dinaciclib (MCL1 reduction). This combination synergistically induced apoptosis in cell lines and primary MCL cells and led to almost complete inhibition of tumour progression in a mouse model. Apoptosis was NOXA-dependent and correlated with the NOXA/MCL1 ratio, highlighting the importance of the NOXA/MCL1 balance for effective cell death induction in MCL.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma.

Role of cyclin dependent kinase 9(CDK9) as a potential target in esophageal adenocarcinoma (EAC) is unknown. We investigated CDK9 protein expression in EAC and Barrett's esophagus and role of CDK9 in oncogenic processes of EAC in vitro and in murine xenografts. The CDK9 expression was significantly higher in EAC as compared to Barrett's esophagus in patient samples. Stable shCDK9 in SKGT4 reduced proliferation by 37% at day 4, increased apoptosis at 48 hours and induced G1 cell cycle arrest at 48 hours (58.4% vs. 45.8%) compared to controls SKGT4 cells. SKGT4-shCDK9 cell-derived tumors were significantly smaller than control SKGT4-derived tumors in xenografts (72.89mm3 vs. 270mm3). Pharmaceutical inhibition of CDK9 by Flavopiridol (0.1μm for 48 hours) and CAN508 (20 and 40μm for 72 hours) induced significant reduction in proliferation and 2-fold increase in apoptosis in SKGT4, FLO1 and OE33 cells. In xenograft models, CAN508 (60 mg/kg/dayx10 days) and Flavopiridol (4mg/kg/dayx10 days) caused 50.8% and 63.1% reduction in xenograft tumors as compared to control on post-treatment day 21. Reduction of MCL-1 and phosphorylated RNA polymerase II was observed with transient shCDK9 in SKGT4 cells but not with stable shCDK9. CAN508 (20 and 40 μm) and Flavopiridol (0.1, 0.2 and 0.3 μm) for 4 hours showed reduction in MCL-1 mRNA (84% and 96%) and protein. Mcl-1 overexpression conferred resistance to Flavopiridol (0.2 μm or 0.4 μm for 48 hours) and CAN 508 (20 or 40μm for 72 hours). Chromatin immunoprecipitation demonstrated significant reduction of binding of transcriptional factor HIF-1α to MCL-1 promoter in FLO-1 cells by CDK9 inhibitors.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/− stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells

Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

Protein Kinase Inhibitor H89 Enhances the Activity of Pseudomonas Exotoxin A-Based Immunotoxins.

HA22 (Moxetumomab pasudotox) is a recombinant immunotoxin (RIT), composed of an anti-CD22 Fv fused to a truncated portion of Pseudomonas exotoxin A. HA22 is in clinical trials to treat patients with hairy cell leukemia and acute lymphoblastic leukemia (ALL). LMB-11 is an improved variant of HA22 with reduced immunogenicity, has a longer half-life in the blood and high activity in vitro and in a Burkitt lymphoma model in vivo Searching for RIT enhancing combination therapies, we found the protein kinase A inhibitor H89 to enhance LMB-11 and HA22 activity 5- to 10-fold on ALL cell lines and on patient-derived ALL samples. In addition, H89 increased the activity of mesothelin-targeting RITs SS1P (38-fold) and RG7787 (7-fold) against the cervical cancer cell line KB31. Unexpectedly we found that the enhancement by H89 was not because of inhibition of protein kinase A; it was partially recapitulated by inhibition of S6K1, which led to inactivation of its downstream targets rpS6 and GSK3β, resulting in a fall in MCL1 levels. H89 increased the rate of ADP-ribosylation of eukaryotic elongation factor 2, enhancing the arrest of protein synthesis and the reduction of MCL1 in synergy with the RIT. In summary, H89 increased RIT activity by enhancing the two key events: ADP-ribosylation of eEF2 and reduction of MCL1 levels. Significant enhancement was seen with both CD22- and mesothelin-targeting RITs, indicating that H89 might be a potent addition to RIT treatment of CD22-positive ALL and mesothelin-expressing solid tumors. Mol Cancer Ther; 15(5); 1053-62. ©2016 AACR.