Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells.Experimental Design: Using samples from patients (n ¼ 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes.Results: AMG-176 incubations resulted in time-and dosedependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high b2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176.Conclusions: AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic.

Inflammatory breast cancer (IBC) is a rare and aggressive presentation of invasive breast cancer with a 62% to 68% 5-year survival rate. It is the most lethal form of breast cancer, and early recognition and treatment is important for patient survival. Like non-inflammatory breast cancer, IBC comprises multiple subtypes, with the triple-negative subtype being overrepresented. Although the current multimodality treatment regime of anthracycline- and taxane-based neoadjuvant therapy, surgery, and radiotherapy has improved the outcome of patients with triple-negative IBC, overall survival continues to be worse than in patients with non-inflammatory locally advanced breast cancer. Translation of new therapies into the clinics to successfully treat IBC has been poor, in part because of the lack of in vitro preclinical models that can accurately predict the response of the original tumor to therapy. We report the generation of a preclinical IBC patient-derived xenograft (PDX)-derived ex vivo (PDXEx) model and show that it closely replicates the tissue architecture of the original PDX tumor harvested from mice. The gene expression profile of our IBC PDXEx model had a high degree of correlation to that of the original tumor. This suggests that the process of generating the PDXEx model did not significantly alter the molecular signature of the original tumor. We demonstrate a high degree of similarity in drug response profile between a PDX mouse model and our PDXEx model generated from the same original PDX tumor tissue and treated with the same panel of drugs, indicating that our PDXEx model had high predictive value in identifying effective tumor-specific therapies. Finally, we used our PDXEx model as a platform for a robotic-based high-throughput drug screen of a 386-drug anti-cancer compound library. The top candidates identified from this drug screen all demonstrated greater therapeutic efficacy than the standard-of-care drugs used in the clinic to treat triple-negative IBC, doxorubicin and paclitaxel. Our PDXEx model is simple, and we are confident that it can be incorporated into a PDX mouse system for use as a first-pass screening platform. This will permit the identification of effective tumor-specific therapies with high predictive value in a resource-, time-, and cost-efficient manner.

Purpose: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases.Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo.Results: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentosephosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma.Conclusions: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.

Background Poor outcomes for patients with ovarian cancer relate to dormant, drug‐resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. Methods By using unbiased small‐interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small‐molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP‐binding protein Di‐Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib‐induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short‐hairpin RNA knockdown of autophagic proteins, and small‐molecule inhibitors of STAT3 and BCL‐2. Results Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3‐expressing tumors treated with crizotinib had significantly decreased tumor burden and long‐term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy‐mediated apoptosis by decreasing phosphorylated STAT3 and BCL‐2 signaling. Conclusions Crizotinib may eliminate dormant, autophagic, drug‐resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second‐look operations should be seriously considered.

Background: Triple-negative breast cancer (TNBC), which affects over 170 000 women worldwide every year, is considered the most arduous to treat subtype of breast cancer. With no targeted therapy, high rates of drug resistance and rapid metastasis, TNBC carries a poor prognosis. The MEK-ERK-MAPK signaling cascade is known to play a role in numerous cancers. Despite the lack of activating Ras/MAPK mutations in breast cancer, transcriptional signatures of this pathway are prevalent in TNBC. Our previous work showed that TNBC patients with tumors overexpressing ERK2 had a lower overall survival rate than did patients with low-ERK2-expressing tumors. MEK inhibitors selumetinib (AZD6244) and pimasertib (AS703026) are active in preclinical models, but not as single agents in the clinic. Using a synthetic lethal siRNA screen, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers. It is associated with cell immortalization, transformation, and chemoresistance. Patients with TNBC tumors expressing high levels of Mcl-1 have lower overall survival and distant-metastasis-free survival rates. We hypothesized that Mcl-1 promotes MEK inhibitor resistance in TNBC. Methods/Results: To model MEK inhibitor resistance, we established selumetinib- and pimasertib-resistant clones of SUM-149 and MDA-MB-231 TNBC cells by continuous exposure to increasing concentrations of inhibitors over a six month period. We confirmed the onset of MEK resistance by demonstrating that resistant cells, in comparison to the parental cells, exhibited no change in cell proliferation upon treatment with the MEK inhibitors. Resistant cells also displayed more effective cell migration and mammosphere formation than parental cells, suggesting a higher fraction of tumor-initiating cells. We found Mcl-1 to be highly expressed in 83% (15 of 18) of TNBC cell lines but only 30% (3 of 10) of other breast cancer cell lines. Resistant cells had higher levels of Mcl-1 than did parental cells. To determine whether Mcl-1 is required for MEK sensitivity, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. The Mcl-1 inhibitor restored MEK sensitivity in both resistant cell lines. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. Similar studies were done using an siRNA against Mcl-1. Conclusion: Our data demonstrate that Mcl-1 may promote TNBC resistance to MEK inhibitors and that Mcl-1 is a promising target for combination therapy. We will continue to explore the mechanisms of MEK inhibitor resistance by screening for additional genes/pathways involved. Our long-term goal is to design rational combination approaches to counteract the emergence of resistance by using novel molecularly targeted therapeutics. Citation Format: Gagliardi M, Chauhan G, Pitner MK, Iles L, Qi Y, Pusztai L, Tripathy D, Bartholomeusz G, Bartholomeusz C. Overcoming MEK inhibitor resistance in triple-negative breast cancer by targeting myeloid cell leukemia-1 (MCL1), an anti-apoptotic protein [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-06.