Guojing Zhang

Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy

The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small-molecule Bcl2-BH4 domain antagonist, BDA-366, that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival molecule to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition upregulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome.

Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment

The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. The current study has demonstrated that Met amplification and hyperactivation is a resistance mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed amplified Met gene and hyperactivated Met, and were cross-resistant to AZD9291 or erlotinib. Met inhibition overcame the resistance of these cell lines to AZD9291 both in vitro and in vivo, including enhancement of apoptosis or G1 cell cycle arrest. Hence, we suggest that Met inhibition is also an effective strategy to overcome resistance of certain EGFR-mutated NSCLCs with Met amplification to AZD9291, warranting the further clinical validation of our findings.

Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer.

Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single‐agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA‐PKcs protein modulation. Gene expression profiling identified differential expression of a 5‐gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell‐specific manner. This potentiation correlates with platinum sensitivity, DNA‐PKcs expression and a 5‐gene expression profile.

Enhancing therapeutic efficacy of the MEK inhibitor, MEK162, by blocking autophagy or inhibiting PI3K/Akt signaling in human lung cancer cells

Human non-small cell lung cancer (NSCLC) displays activated MEK/ERK signaling due to a high frequency of K-Ras mutation and is thus a potential candidate for MEK-targeted therapy. The current study focuses on demonstrating the activity of MEK162 (binimetinib), a MEK inhibitor under clinical testing, against NSCLC and exploring possible mechanism-driven strategies to enhance its therapeutic efficacy. MEK162 inhibits the growth of human NSCLC cell lines with varied potencies through induction of G1 cell cycle arrest and apoptosis. Moreover, it induces autophagy and accordingly the combination of MEK162 with the autophagy inhibitor, chloroquine, synergistically inhibits the growth of NSCLC cells and enhances apoptosis. MEK162 activates Akt signaling while effectively inhibiting MEK/ERK signaling. Accordingly, the combination of MEK162 and BKM120 (buparlisib), a pan-PI3K inhibitor, abrogates induced Akt activation and significantly augments therapeutic efficacy against the growth of NSCLC cells both in vitro and in vivo. Hence our findings warrant further evaluation of these rational combinations in the clinic.

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 vivo. Conclusions: 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.

Anaplastic lymphoma kinase (ALK) gene alteration in signet ring cell carcinoma of the gastrointestinal tract

Objectives: ALK-EML4 translocation is an established driver aberration in non-small cell lung cancer (NSCLC), with reported predilection for cases with signet ring histology. We assessed the presence of anaplastic lymphoma kinase (ALK) gene rearrangements in signet ring cancers arising in the stomach and colon. Methods: Histologically confirmed cases of signet ring adenocarcinoma of the stomach or the colon were identified. The presence of the classic ALK and EML4 fusion gene was initially determined by fluorescence in-situ hybridization (FISH) technique. Immunohistochemistry (IHC) was performed using two previously validated antibodies, ALK1 clone (1:100; DAKO) and 5A4 (Novocastra, Leica Biosystems) along with positive controls of ALK-translocated lung cancer. Results: We employed 42 cases of signet ring carcinoma diagnosed between 2001 and 2011; 25 gastric and 17 colon cancer. Median age 63.3 years; male/female 17/25; race, black 47.5%, white 47.5%, others, 5%; stage I, 21.4%; stage II, 31%; stage III, 26.2%; stage IV, 21.4%. One of 42 cases (2.3%) was positive for ALK translocation by FISH using the standard criteria of at least 15% positive cells for the break-apart signal (50–70 cells enumerated per case). Using a less restrictive cut-off of 10% positive cells, 7 cases (16%) were considered possibly positive. None of the ‘possibly positive’ cases was found to harbor ALK translocation by another molecular testing approach (IHC). IHC with two previously validated monoclonal antibodies showed 0 of 42 (0%) cases positive. Conclusions: ALK gene rearrangement is very rare in gastrointestinal cancers and enrichment strategy focusing on signet ring cell histology did not significantly improve the detection rate.

MAST1 Drives Cisplatin Resistance in Human Cancers by Rewiring cRaf-Independent MEK Activation

Platinum-based chemotherapeutics represent a mainstay of cancer therapy, but resistance limits their curative potential. Through a kinome RNAi screen, we identified microtubule-associated serine/threonine kinase 1 (MAST1) as a main driver of cisplatin resistance in human cancers. Mechanistically, cisplatin but no other DNA-damaging agents inhibit the MAPK pathway by dissociating cRaf from MEK1, while MAST1 replaces cRaf to reactivate the MAPK pathway in a cRaf-independent manner. We show clinical evidence that expression of MAST1, both initial and cisplatin-induced, contributes to platinum resistance and worse clinical outcome. Targeting MAST1 with lestaurtinib, a recently identified MAST1 inhibitor, restores cisplatin sensitivity, leading to the synergistic attenuation of cancer cell proliferation and tumor growth in human cancer cells and patient-derived xenograft models.

Modulation of Bax and mTOR for Cancer Therapeutics

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR.

Abstract 2105: c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors

c-Met amplification and acquisition of a second T790M mutation are key mechanisms accounting for majority of resistant cases to first generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs; i.e., erlotinib). The third generation EGFR-TKIs (e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants while sparing wild-type EGFR, represent very promising therapeutic options for NSCLC patients who have become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. We show that c-Met amplification and hyperactivation is an universal mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed elevated levels of c-Met (due to gene amplification) and p-c-Met and were cross-resistant to AZD9291 or erlotinib. Both chemical and genetic inhibition of c-Met overcame the resistance of these cell lines to AZD9291 including enhancement of apoptosis or G1 cell cycle arrest. Consistently the combination of AZD9291 and c-Met inhibition effectively inhibited the growth of both erlotinib- and AZD9291-resistant HCC827 xenografts in nude mice. Hence, we suggest that inhibition of c-Met is also an effective strategy to overcome resistance of EGFR-mutated NSCLCs with c-Met amplification or hyperactivation to AZD9291, providing the rationale for clinical development of this novel combination strategy. (SSR, TKO and SYS are Georgia Research Alliance Distinguished Cancer Scientists) Citation Format: Puyu Shi, You-Take Oh, Guojing Zhang, Weilong Yao, Ping Yue, Rajani Kanteti, Jacob Riehm, Ravi Salgia, Taofeek Owonikoko, Suresh S. Ramalingam, Mingwei Chen, Shi-Yong Sun. c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2105.

Abstract 922: Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Molecular characterization of small cell lung cancer (SCLC) can lead to improved outcome through the identification of tumor subtypes with differential clinical outcome and response to treatment. We characterized SCLC cell lines and patient samples by a combination of a genome-wide and a custom expression panel to assess expression changes associated with cisplatin sensitivity, in the case of cell lines, and overall survival, in terms of SCLC patients.Method: SCLC cell lines (H69, H128, H146, H526, H187, H209, D53, DMS153, and DMS114) and 79 archival samples of pulmonary neuroendocrine tumor samples were employed. Sensitivity to cisplatin, and PARP inhibitor, veliparib was determined by MTS assay. In order to identify expression profiles characteristic of sensitive and insensitive cell lines, gene expression for untreated and treated cells was obtained from Illumina microarray gene expression and NanoString platforms. Comparisons of expression were performed between treated cell lines and controls using a combination of ANOVA and empirical variance distributions to assess sensitivity. Prognostic impact of differentially expressed genes in cell lines was assessed in patients using gene expression obtained by NanoString and protein expression by immunohistochemistry (IHC). Kaplan Meier Method, Logrank test, and Cox proportional hazard model were employed for testing differences in survival. Additionally, a quantile survival analyses was performed to identify patient subgroups associated with early and/or late changes in expression.Results: Unsupervised analysis of the Illumina probesets identified a 23 gene panel with differential expression between cell lines with differing platinum sensitivity: Sensitive - GLS, UBEC2, HACL1, MSI2 and LOC100129585; insensitive - CENPE, CRYGS, FAM83D, FLJ44342, GNA12, LOC88523, LRDD, N4BP2L2, SLC35A3, SPC25; shared - AURKA, CENPA, DLGAP5, HMMR, KIF20B, LOC100129585, LOC100131735, RBMX, SFRS3. Unsupervised analysis of expression data from Illumina and nCounter NanoString showed considerable concordance in cluster patterns. The Cox regression models of gene expression on overall survival identified LOC100131735 as a predictive marker and, SLC35A3, SPC25, DLGAP5 and UBE2C genes as prognostic biomarkers. Concomitantly high expression of SLC35A3 and GLS genes characterized SCLC with prolonged survival (Logrank p=0.025) while a trend toward reduced risk of death (HR: 0.798; 95%CI, 0.375-1.700, p=0.559) was observed with high GLS protein expression.Conclusions: Our use of both cell line and patient SCLC data has provided evidence of markers characteristics of cisplatin insensitivity that also show a relation with poor overall survival in patients. Additional studies to fully understand the mechanisms underlying such markers are currently underway. Supported through the Georgia Cancer Coalition Distinguished Scholar Award and NIH 5K23CA164015 grant to TK Owonikoko Citation Format: Taofeek Kunle Owonikoko, Guojing Zhang, Gabriel L. Sica, Zhengjia Chen, Jeffrey M. Switchenko, Sungjin Kim, Anthony A. Gal, Suresh S. Ramalingam, Xingming Deng, Michael R. Rossi, Jeanne Kowalski, Fadlo R. Khuri. Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 922. doi:10.1158/1538-7445.AM2014-922