Lapatinib Resistant Research Articles

Abstract LB228: Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells

Abstract Background: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinases, which is approved for HER2-positive metastatic breast cancer as second line treatment. Significant proportion of patients experience disease progression due to acquired resistance. Activation of DNA damage repair (DDR) is one of the drug-resistance mechanism, however, the impact of DDR on sensitivity to lapatinib is unclear. Thus, lapatinib resistance mechanism was explored from the standpoint of DDR activation. Methods: Acquired lapatinib-resistant (LR) SK-BR-3 cell lines were generated by continuously exposing to lapatinib, starting with 30 nmol/L and incrementally increasing to 10 μmol/L over 7 months. MTT assay was used to confirm lapatinib sensitivity. Cell cycle progression was analyzed using BrdU assay. Replication fork speed and stalled fork were analyzed by DNA fiber assay. Expression of the molecules was examined using Western blotting, immunofluorescence assay and transcriptome data analysis. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. Results: G1/S phase transition was increased and early/late S phase population was increased in SK-BR-3 LR cells. Replication fork speed was accelerated and replication stress are elevated in SK-BR-3 LR cells. p-Chk1, Rad51, Rad51B, Rad51C and XRCC3 were upregulated in SK-BR-3 LR cells. After irradiation or treatment with ATR inhibitor, γ-H2AX was continuously increased in parental cells. In contrast, γ-H2AX did not change significantly in SK-BR-3 LR cells, and Chk1 was activated after irradiation or treatment with ATR inhibitor. Tail of comet disappeared at early time point in in SK-BR-3 LR cells after induction of DNA damage compared with parental cells. These results demonstrated that DDR was activated and DNA damage repair capacity was enhanced in LR cells. Conclusion: Replication stress is elevated in SK-BR-3 LR cells. Up-regulation of molecules involved in the homologous recombination repair pathway was observed in SK-BR-3 LR cells. Moreover, DNA repair capacity was increased in SK-BR-3 LR cells. These data suggested that activation of DNA damage repair pathway caused by replication stress contributes to lapatinib resistance. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, Changyun Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB228.

Novel 6, 7-disubstituted 7H-purine analogues as potential EGFR/HER2 dual kinase inhibitors overcome lapatinib resistance: Design, synthesis, in-vitro and in-vivo evaluation

We synthesized and evaluated a new series of 6, 7 disubstituted-7H-purine derivatives (10a-u) that inhibit cancer growth via dual inhibition of EGFR/HER2. A total of twenty-one compounds were synthesized employing effective synthetic strategies and characterized. Initially, all compounds were screened in-vitro for kinase inhibition against a panel of seven kinases, including four members of the EGFR family, VEGFR, PDGFRα, and PDFGRβ. Five compounds from three synthetic series, 10d, 10e, 10i, 10 m, and 10r, were identified as potent EGFR family kinase inhibitors. Compound 10r effectively inhibited EGFR/HER2 dual kinases with an IC50 of 0.017 ± 0.003/0.014 ± 0.002 µM. In addition, in-vitro cytotoxic data indicated that compounds 10i and 10r significantly altered the growth, resulting in potential anti-proliferation in a panel of breast cancer cells except for MCF-7. Following the findings of kinase inhibition, 10r demonstrated the lowest growth inhibition (IC50) against all breast cancer cells, ranging from 1.537 to 3.196 µM, except MCF-7 cells. Interestingly, 10i and 10r showed promising IC50 ranging from 1.682 to 2.236 µM against the Lapatinib resistant lines BT-474/L and SKBR-3/L. Furthermore, apoptotic analysis of 10r revealed that the most significant percentage of cells, including Lapatinib-sensitive and resistant BT-474 and SKBR-3 cells, were forced into the accelerated death phase over 10r treatment, indicating effective apoptotic behaviour. The selective downregulation of dual EGFR/HER2 and their downstream signaling regulators ERK1/2 and AKT were observed over the treatment of 10r in both BT-474/L SKBR-3/L cells. Inconsistent with the in-vitro effects, 10r exhibited the same result in BT-474 and BT-474/L xenograft mice models. Finally, molecular docking studies revealed that 10r strongly binds to EGFR and HER2 via 'H' and 'pi' bond interactions, resulting in irreversible dual kinase inhibition. Thus our findings show that substituting 6, 7 positions in the purine core might result in potential anti-cancer agents, including combating the ability of Lapatinib resistance.

Strategic co-targeting of FOXM1 and EGFR-ERBB2 signaling in ovarian cancer

Objectives: Epithelial ovarian cancer (EOC) is a challenging disease due to chemoresistance, and short-term recurrence. Chemoresistance to anti-cancer drugs substantially reduces survival in EOC. In this study, we showed that chemoresistance to lapatinib (a dual EGFR-ERBB2 kinase inhibitor) and thiostrepton (FOXM1 inhibitor), induced the epithelial-mesenchymal transition and stemness in OC cells. Aberrant expression of FOXM1 and EGFR-ERBB2 proteins are common in many solid cancers including serous EOC. However, the precise mechanism of FOXM1, EGFR-ERBB2 in the chemoresistance of OC remains unclear. Methods: We blocked FOXM1 and EGFR-ERBB2 signaling by either genetically or pharmacologically, then evaluated the effects of thiostrepton and lapatinib on OC proliferation, cell cycle changes, and migratory effects by trypan blue exclusion, 3-D spheroid formation ability, growth assays, confocal microscopy, cell cycle, apoptosis analysis, and in vivo tumor growth assay. Results: In our study, we identified the mechanism of how tumor cells relieve their dependency on EGFR-ERBB2 signaling and adopt alternative and sustainable signaling mechanisms like FOXM1 for resistance to ERBB-therapy and metastasis. To characterize the mechanism of lapatinib and thiostrepton resistance in OC cells, we developed lapatinib and thiostrepton resistant OC cells. We observed that long-term treatment of lapatinib decreased, the levels of pEGFR (Y1068) and pERBB2 (Y1248) proteins, whereas the levels of both phosphorylated and total protein of CDK1 and pPLK1 were upregulated. Strikingly, we found that FOXM1 level is upregulated in the cells are resistant to lapatinib. Next, we determined the nuclear level of FOXM1 which is important for the transcriptional activation of oncogenes. Importantly, we found an increased localization of FOXM1 in the nucleus in lapatinib resistant cells. In contrast, we found that the thiostrepton resistant OC cells express high levels of EGFR and ERBB2. In conjunction, we found that combined FOXM1 and EGFR-ERBB2 inhibition by the sub-lethal concentration of thiostrepton and lapatinib treatment reduced EGFR-ERBB2 and FOXM1 expression and induced apoptosis and inhibition of the colony-forming ability of tumor cells more than a single agent treatment. In addition, our Western blots, immunofluorescence, and immunohistochemistry assays in combination with in vivo studies demonstrated that the combined pharmacological inhibition of FOXM1 by thiostrepton reduced nuclear expression of the FOXM1 in lapatinib resistant cells while EGFR-ERBB2 inhibitor lapatinib downregulates the EGFR-ERBB2 in thiostrepton resistant cells. Taken together, our data demonstrated that the lapatinib resistant mechanism operates through FOXM1 activation likely through CDK1 or PLK1. To inhibit the levels of FOXM1, we used thiostrepton, which is capable of inhibiting the levels of FOXM1 in OC cells, and the cells which are resistant to thiostrepton express high levels of p-EGFR, p-ERBB2, and p-ERK2. In support to our results, we found that thiostrepton resistant cells express high levels of pEGFR and pERBB2, as well as high levels of EGF mRNA. Conclusions: Taken together, our data demonstrated that FOXM1 and EGFR-ERBB2 expression and activation provide a compensatory mechanism against EGFR/ERBB2 or FOXM1 therapy. Thus, inhibiting FOXM1 and EGFR/ERBB2 levels would provide an opportunity to sensitize OC cells resistant to FOXM1 or EGFR/ERBB2 therapy in OC patients.

Abstract PS16-28: Runx3 regulates emt during development lapatinib resistance

Abstract RUNX3 regulates EMT during development lapatinib resistanceBackground: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinase, which is used for HER2 positive metastatic breast cancer patients. However, significant proportion of patients relapse due to acquired resistance. The epithelial to mesenchymal transition (EMT), which induces cell migration, invasion and cancer stem cell progression, was one of the mechanism that confers to drug resistance. We determined the correlation of lapatinib resistance and EMT, and identified a regulatory factor of EMT which associated with the development process of acquired lapatinib resistance.Methods: Acquired lapatinib resistant SK-BR-3 (SK-BR-3 LR) cells were established by continuously exposing to lapatinib for 7 months. The sensitivity of lapatinib was confirmed by MTT assay. Cell cycle progression was verified using flow cytometry analysis. Expression of signal transduction molecules were determined using quantitative PCR, western blotting and transcriptome data analysis. Cell migration and invasion ability were verified using wound healing assay and Boyden chamber assay. siRNA knock-down system were used for further analysis. Results: Lapatinib resistant (SK-BR-3 LR) cells showed aggressive morphology compared with parental cells. The growth rate and cell cycle progression were increased in SK-BR-3 LR cells. Expression of Snail, Vimentin, SOX2, Nanog, TGF-b1 and Smad proteins increased in SK-BR-3 LR cells. In SK-BR-3 LR cells, cell migration and invasion were significantly increased. Correlated with a promoter methylation of RUNX3, expression of RUNX3 was down-regulated in SK-BR-3 LR cells. In Runx3 knock-down cells TGF-b1, SOX2, smad molecules were up-regulated. Conclusion: SK-BR-3 LR cells showed aggressive phenotype. Expression of EMT markers, cancer stem cell markers, TGF-β pathway associated molecules and Smad proteins were increased in SK-BR-3 LR cells. Also, the increase of cell migration and invasion was observed in LR cells. RUNX3 affected to cell migration and invasion through regulation of EMT associated molecules. Therefore, RUNX3 might be a specified molecule, which partially contributes resistance to lapatinib through regulates EMT. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yoonjung Park, Yu Jin Kim, Seock-Ah Im. Runx3 regulates emt during development lapatinib resistance [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-28.

Rational Design Explore Trifluoromethyl Placement on 6, 7-Disubstituted-7h-Purine Scaffold Potentially Induces Apoptosis Through Dual Inhibition of EGFR/HER2 in Breast Cancers Irrespective of Lapatinib Resistance

A new series of 6, 7 disubstituted-7H-purine derivatives (10a-u) were synthesized and evaluated for their anticancer activity. Using simple and efficient methodologies, 21 compounds were prepared and characterized. We screened the compounds in-vitro to derive conclusions about their kinase inhibition against a panel of seven kinases including EGFR family, VEGFR, PDGFRα, and PDFGRβ. Five compounds 10d, 10e, 10i, 10m , and 10r from three synthetic series, identified as potent EGFR family kinase inhibitors. Compound 10r (Trifluoromethyl substituted) displayed potent dual inhibition of EGFR/HER2 with a kinase inhibition (IC 50 ) of 0.017 ± 0.003/0.014 ± 0.002 µM. Cell cytotoxicity of all molecules was evaluated against a panel of breast cancer cells having a heterogeneous expression of EGFR and HER2. In-vitro cytotoxic results revealed that compounds 10i, and 10r significantly altered the growth which finally leads to potential anti-proliferation in selected cancer cells except for MCF-7 when compared with Lapatinib. Following kinase inhibition results, 10r showed the lowest growth inhibition IC 50 against all breast cancer cells ranging from 0.098 to 0.136 µM except MCF-7 cells. Based on potential cytotoxic studies compounds 10i and 10r were successfully evaluated against Lapatinib resistant lines BT-474/L and SK-BR3/L and showed promising IC 50 ranging from 0.096 to 0.125 µM. Further apoptotic evaluation of 10r showed the highest percentage of cells including both sensitive and resistant BT-474, and SK-BR3 cells driven into accelerated death phase which indicated the strength of newly designed molecular series towards cytotoxic and apoptotic behaviour. Molecular docking studies revealed that 10r successfully binds to both EGFR and HER2 with ‘H’ and pi bond involved interactions which brings irreversible dual inhibition of kinases. Our study suggests that substitution on 6, 7 positions, in the purine core would generate potential anti-cancer agents including combating the ability of Lapatinib resistance.

50P Efficacy and safety analysis of pyrotinib in lapatinib resistant HER2-positive metastatic breast cancer: A retrospective study

Lapatinib has shown effectiveness in treating HER2-positive metastatic breast cancer, but therapies after lapatinib resistance are still controversial. In this retrospective study, we assessed the efficacy and safety of pyrotinib in lapatinib resistant HER2-positive metastatic breast cancer. From August 2018 to March 2020, 76 HER2-positive metastatic breast cancer patients who previously failed by lapatinib received pyrotinib in four hospitals. The primary endpoint was investigator-assessed progression-free survival (PFS) per Respond Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The secondary endpoint was the overall survival (OS) and safety of pyrotinib. 66 (86.8%) patients received pyrotinib immediately after lapatinib and 10 (13.2%) received pyrotinib following one or more other therapies. The median PFS of pyrotinib was 8.0 months (95%CI 5.1-10.9) and OS has not reached. Objective response rate (ORR) was 17.1%, and clinical benefit rate (CBR) was 60.5%. Patients who benefited from lapatinib ≥6.0 months were found to have a longer PFS (P=0.034; stratified hazard ratio [HR] 0.534, 95%CI 0.293-0.975). In patients who had received lapatinib in 3 or later line therapy (35, 46.1%), the median PFS of pyrotinib was 9.9 months (95%CI 6.97-12.83) and was relevant to whether lapatinib PFS had reached 6.0 months (P=0.044; HR 0.412, 95%CI 0.167-1.013). No relations were detected between pyrotinib PFS and estrogen receptor (ER) status, trastuzumab resistance, brain metastasis or the sequential use of pyrotinib. In patients who had received lapatinib earlier (41,53.9%), the median PFS of pyrotinib was 6.4 months (95%CI 3.57-9.23). No relevant factors were observed. There was no difference in PFS between these two groups with different lapatinib lines. Toxicity profiles were similar in both groups. The most common adverse effects were diarrhea (34, 44.7%) and hand-foot syndrome (10, 13.2%). Pyrotinib could improve the survival of HER2-positive metastatic breast cancer patients after the failure of lapatinib. For patients who benefited from lapatinib ≥ 6.0 months in 3 or later line therapy, pyrotinib could provide a clinically meaningful longer PFS.

Abstract 1916: Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Abstract Background: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). EAC overexpresses HER2 and lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways fails to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of Warburg effect in lapatinib resistance mechanism of EAC cells. Methods: First we established lapatinib-resistant OE19 (LPR-OE19) cells from OE19 EAC cells and characterized it. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs. We then explored the antitumor efficacy following lapatinib and shikonin mono and combination therapies in murine subcutaneous xenograft model of human EAC. Results: Lapatinib-resistant OE19 (LPR-OE19) cells were established from OE19 cells by intermittent exposure to increasing concentrations of lapatinib for a period of total six months. Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels whereas showed upregulation of PKM2 only at protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. More interestingly, knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 bound to molecular chaperone HSP40. In subcutaneous xenografts using LPR-OE19 cells, average net tumor growth after two weeks in different therapy groups was 494.72 mm3 in control, 403.11 mm3 after lapatinib (p=0.241), 339.48 mm3 after shikonin (p=0.0478), and 141.59 mm3 after shikonin plus lapatinib (p=0.0003). Conclusions: In conclusion, these data suggest that combination therapy with lapatinib and glycolytic inhibitor shikonin should be tested as a potent option for lapatinib resistant HER2-positive EAC patients and could be a novel treatment strategy for specific EAC. In addition, it is suggested that targeting HSP40-PKM2 interaction as an innovative therapeutic approach to overcome lapatinib resistance in EAC. Citation Format: Md Sazzad Hassan, Samantha Holmes, Victoria Makuru, Chloe Johnson, Urs von Holzen. Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1916.

Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation

BackgroundChemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance.MethodsSulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay.ResultsTo explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666–1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4−/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells.ConclusionCollectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

Targeting the Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) overexpresses HER2. Lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways failed to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of the Warburg effect in the lapatinib resistance mechanism of EAC cells.
 Methods: First we established a lapatinibresistant OE19 (LPR-OE19) cell line from OE19 EAC cells. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs.
 Results: Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels, but showed upregulation of PKM2 only at the protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. Knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition, and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 binds to molecular chaperone HSP40.
 Conclusions: These data suggest that targeting HSP40PKM2 interaction could be an innovative therapeutic approach to overcome lapatinib resistance in EAC.

Abstract 289: MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma

Abstract Introduction: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). Unfortunately, patients treated with lapatinib, a dual EGFR and HER2 inhibitor, frequently develop resistance. Lapatinib fails to improve patient survival in HER2-postive EAC, and the mechanisms contributing to resistance remain largely unknown. Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset EAC. In this study, we therefore studied the role of MET activation in lapatinib resistance mechanisms in experimental EAC. Methods: We first characterized MET and HER2 activation in a panel of human EAC cell lines, and the differential susceptibility of these EAC cell lines to single agent or combination of MET and HER2 inhibitions by foretinib and lapatinib, respectively. We then explored the antitumor efficacy with survival advantage following foretinib and lapatinib monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. We also tested MET and HER activation status with sensitivity to their inhibitors in newly generated lapatinib resistant OE19 EAC cells (OE19-LPR). Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as single agents. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell proliferation and xenograft tumor growth, induced apoptosis, and significantly enhanced overall mouse survival, thus overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent with lack of enhanced growth inhibition when the two agents were combined. Interestingly, OE19-LPR cells showed significant upregulated expression of phosphorylated MET compared to parent OE19 cells, detected by both Activesignal assay and Western blot analysis. In addition, OE19-LPR cells showed significantly reduced sensitivity to lapatinib compared to parent OE19 cells, and the co-administration of foretinib and lapatinib synergistically inhibited cell proliferation in OE19-LPR cells. Conclusion: These data suggest that resistance to HER2 targeted therapies in HER2 and MET overexpressed EAC cells can be due to MET activation. Thus MET and HER2 targeted therapy appears to be a sensible treatment strategy for HER2 positive MET-overexpressing EAC. Citation Format: Md Sazzad Hassan, Fiona Williams, Lucia Petrova, Niranjan Awasthi, Margaret Schwarz, Roderich Schwarz, Urs von Holzen. MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma [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 289.

Abstract P6-21-14: Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer

Abstract Breast cancer metastasis, when cancer cells move and establish tumors in distant organs, confounds treatment options. Therefore, there is an unmet need for targeted therapeutics to address metastasis, especially in the intractable HER2 positive (+) breast cancer type. HER2 (+) breast cancer is treated with the monoclonal HER2 antibody trastuzumab and the HER2/EGFR targeting small molecule lapatinib. However, ˜20% of early stage breast cancer patients and ˜70% of patients with metastatic disease are resistant to trastuzumab. Trastuzumab-resistant breast cancers circumvent HER2 inhibition via bypass signaling, which includes the PI3-K/Akt/mTOR pathway. HER2/EGFR and PI-3K/Akt signaling activate the Rho GTPases Rac and Cdc42, and overexpression of Rac or its downstream effector p21-activated kinase (PAK) significantly diminishes response to anti HER2 therapy and patient survival. Therefore, targeting Rac and Cdc42, pivotal regulators of cancer cell migration/invasion, and thus, metastasis, is a viable option to overcome HER2/EGFR therapy resistance. First, we designed and developed the Rac inhibitor Ehop-016 (US patents 8,884,006 B2, 9,278,956B1), which inhibits Rac activation with an IC50 of 1 μM and reduces breast cancer growth and metastasis in mouse models of trastuzumab resistant HER2 (+) breast cancer. A more potent dual Rac and Cdc42 inhibitor, MBQ-167 (US Patent 9,981,980 B2), inhibits Rac and Cdc42 activation with IC50s of 103 nM and 78 nM, respectively. In vivo, MBQ-167 inhibits trastuzumab resistant HER2 (+) mammary tumor growth and metastasis by ˜90-100% (Humphries-Bickley et al. 2017, Mol Cancer Therap). Therefore, Rac/Cdc42 inhibition blocks cell proliferation, cell cycle progression, migration, and induces apoptosis to ultimately impede tumor growth and metastasis in trastuzumab resistant HER2 (+) breast cancer models. To further investigate the role of Rac/Cdc42 inhibitors in overcoming HER2 therapy resistance, we developed a lapatinib resistant variant of the SKBR3 HER2 (+) breast cancer cell line and found that Rac was overexpressed and over activated in the therapy resistant variant compared to the parental cells. Accordingly, the Rac/Cdc42 inhibitors overcame lapatinib resistance by decreasing cell viability and inducing apoptosis in parental and SKBR3 therapy resistant variants. Pharmacokinetic analysis in mouse plasma demonstrated that the bioavailability of EHop-016 and MBQ-167 is ˜30% with a half-life of 3-4 h. Therefore, to increase the bioavailability of Rac/Cdc42 inhibitors and to facilitate sensitization of trastuzumab therapy, we designed and developed novel nanoliposomal formulations containing Rac/Cdc42 inhibitors with trastuzumab on the outer surface to target HER2 (+) breast cancer cells. This data demonstrates the utility of developing Rac/Cdc42 inhibitors as mono or combined therapy with current targeted therapeutics for HER2 (+) breast cancer. Citation Format: Rivera-Robles MJ, Medina-Vlazquez JI, Borrero-Garcia LD, Velazquez-Vega LE, Maldonado MdM, Ruiz-Calderon J, Castillo-Pichardo L, Vivas-Mejia P, Dharmawardhane S. Targeting Rac/Cdc42 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer [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 P6-21-14.

Phosphoproteomic analysis reveals PAK2 as a therapeutic target for lapatinib resistance in HER2-positive breast cancer cells

The human epidermal growth factor receptor 2 (HER2)-positive breast cancer with overexpression of HER2 accounts for approximately 25% of breast cancers and is more aggressive than other types of breast cancer. Lapatinib has been widely used as a HER2-targeted therapy, however, a number of patients develop lapatinib resistance and still suffer from poor prognosis. Therefore, it is essential to identify novel therapeutic targets that could overcome lapatinib resistance. In this study, we carried out phosphoproteomic analysis of lapatinib sensitive and resistant cell lines (SKBR3 and SKBR3-LR) using stable isotope labeling with amino acids in cell culture (SILAC). We identified 3808 phosphopeptides from 1807 proteins and then analyzed signaling pathways, Gene Ontology, and protein-protein interaction networks. Finally, we identified PAK2 as a therapeutic target from the network analysis and validated that PAK2 knockdown and PAK inhibitor treatment resensitize the lapatinib resistant cells to lapatinib. This results suggest that PAK2 is a potent therapeutic target to overcome acquired lapatinib resistance in HER2-positive breast cancer cells.

Pre-clinical evaluation of the PP2A inhibitor LB-100 for the treatment of lapatinib resistant HER2-positive breast cancer.

e13021Background: We have previously shown that protein phosphatase 2A (PP2A) may mediate acquired lapatinib resistance and inhibition of PP2A with okadaic acid enhances lapatinib response in vitro...

Structural investigations on mechanism of lapatinib resistance caused by HER-2 mutants.

HER-2 belongs to the human epidermal growth factor receptor (HER) family. Via different signal transduction pathways, HER-2 regulates normal cell proliferation, survival, and differentiation. Recently, it was reported that MCF10A, BT474, and MDA-MB-231 cells bearing the HER2 K753E mutation were resistant to lapatinib. Present study revealed that HER-2 mutant K753E showed some contrasting behaviour as compared to wild, L768S and V773L HER-2 in complex with lapatinib while similar to previously known lapatinib resistant L755S HER-2 mutant. Lapatinib showed stable but reverse orientation in binding site of K753E and the highest binding energy among studied HER2-lapatinib complexes but slightly lesser than L755S mutant. Results indicate that K753E has similar profile as L755S mutant for lapatinib. The interacting residues were also found different from other three studied forms as revealed by free energy decomposition and ligplot analysis.

Activation of cancerous inhibitor of PP2A (CIP2A) contributes to lapatinib resistance through induction of CIP2A-Akt feedback loop in ErbB2-positive breast cancer cells.

Lapatinib, a small molecule ErbB2/EGFR inhibitor, is FDA-approved for the treatment of metastatic ErbB2-overexpressing breast cancer; however, lapatinib resistance is an emerging clinical challenge. Understanding the molecular mechanisms of lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast cancer. Our study investigated the role of CIP2A in the anti-cancer efficacy of lapatinib in ErbB2-overexpressing breast cancer cells. We found that lapatinib concurrently downregulated CIP2A and receptor tyrosine kinase signaling in ErbB2-overexpressing SKBR3 and 78617 cells; however, these effects were attenuated in lapatinib-resistant (LR) cells. CIP2A overexpression rendered SKBR3 and 78617 cells resistant to lapatinib-induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via lentiviral shRNA enhanced cell sensitivity to lapatinib-induced growth inhibition and apoptosis. Results also suggested that lapatinib downregulated CIP2A through regulation of protein stability. We further demonstrated that lapatinib-induced CIP2A downregulation can be recapitulated by LY294002, suggesting that Akt mediates CIP2A upregulation. Importantly, lapatinib induced differential CIP2A downregulation between parental BT474 and BT474/LR cell lines. Moreover, CIP2A shRNA knockdown significantly sensitized the BT474/LR cells to lapatinib. Collectively, our results demonstrate that CIP2A is a molecular target and resistance factor of lapatinib with a critical role in lapatinib-induced cellular responses, including the inhibition of the CIP2A-Akt feedback loop. Further investigation of lapatinib-mediated CIP2A regulation will advance our understanding of lapatinib-associated anti-tumor activities and drug resistance.

Abstract 4116: Acquired lapatinib resistant induces EMT-phenotype in breast cancer cells via Src and RUNX3

Abstract Background: Lapatinib is an effective EGFR and HER2 targeting small molecular tyrosine kinase inhibitor which is widely used for HER2 positive breast cancer patients. However, patients eventually acquire resistance, limiting its long-term use. Overexpression of heregulin, a HER3 ligand, acquired stemness were mechanism that confers resistance to the anti-HER2 lapatinib. However, still, unknown mechanisms of developing lapatinib resistant remain as a question to solve. Thus, we tried to find out there is a novel mechanism which related to lapatinib resistant, and any specified molecules were involved in this process. Methods: Acquired resistant SK-BR-3 cells were established by chronic exposure to lapatinib or trastuzumab. Lapatinib or trastuzumab sensitivity were confirmed by MTT assay. Western blotting was used to determine signal transduction molecule changes. Wound healing assay and Boyden chamber assay were conducted for verifying invasive ability. Whole exome sequencing (WES) and siRNA knock-down system were used for further analysis. Results: Lapatinib resistant (LR) cell lines showed down-regulation of p-HER2, p-Akt, and p-Erk. The activity of Src family kinase was increased in LR cells. Vimentin, famous EMT marker, is also increased in LR cells. Migration and invasion were significantly increased in LR cells. Correlated with a missense mutation of RUNX3, which identified by WES, expression of RUNX3 was decreased in LR cells. Moreover, siRNA knock-down parental cells showed more resistance to lapatinib. Conclusion: The increase of Src activation, cell migration, and invasion was observed in LR cells. RUNX3, which identified by WES, affected to lapatinib sensitivity in SK-BR-3 cells. Therefore, RUNX3 might be a specified molecule, which partially contributes resistance to lapatinib. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Dong Hyeon Ha, Hyemin Jang, Yu Jin Kim, Kyung-Hun Lee, Tae-Yong Kim, Seock-Ah Im. Acquired lapatinib resistant induces EMT-phenotype in breast cancer cells via Src and RUNX3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2017-4116

Abstract 104: miRNAs in the 14q32 cluster are involved in lapatinib resistance

Abstract HER2 is over-expressed in around 20-25% of breast cancers. Due to increased cell growth and differentiation signaling resulting from HER2 over-expression, this sub-type of the disease is associated with aggressive tumor progression and poor prognosis. HER2 targeted therapy has improved patient prognosis, however, only a subgroup of patients derives the optimal benefit, while other patients have refractory disease or develop resistance. miRNAs are small non coding RNAs involved in the RNAi machinery. They are often aberrantly expressed in tumors and miRNA profiles have been seen to evolve over the course of treatment implicating them in therapeutic resistance. Methods: miRNA array completed on the Human TaqMan miRNA card. Gene expression profiling performed by Illumina Human HT12 v4.0 array. Differential gene expression used LIMMA1 empirical Bayes statistics module. Adjusted p-values calculated by Benjamini and Hochberg procedure. In silico target prediction used miRWalk. Lapatinib resistant cells generated by culturing cells in increasing doses of lapatinib up to 2µM. IC50 obtained from drug cytotoxicity measured by MTT analyzed by nonlinear regression. Results: A panel of miRNAs differentially expressed in paired lapatinib sensitive/resistant BT-474 HER2 positive breast cancer cells were identified by miRNA array. Three miRNAs, located in the 14q32 region, were selected for further investigation of their role in resistance to HER2-targeted therapy: miR-127-3p, miR-409-3p, and miR-495-3p. RT-qPCR also confirmed upregulation of these miRNAs in HCC1954 and SKBR-3 HER2 positive cells with acquired resistance to lapatinib, linking increased miRNA expression with acquired resistance. To understand the mechanism behind the miRNA expression in resistant cells we explored epigenetic changes between the sensitive and resistant lines. Global methylation reversal revealed upregulation of all miRNAs in the sensitive cells with a further increase upon addition of an HDAC inhibitor. De-methylation had less effect in resistant cells, indicating loss of methylation as a key component in miRNA upregulation in the process of lapatinib resistance. Using differential gene expression analysis in paired lapatinib-sensitive/resistant BT-474 cells combined with in silico analysis we identified putative targets of these miRNAs. BASP1, a c-MYC transcriptional inhibitor, was down-regulated in the BT-474/L resistant cells. Knock-down altered sensitivity to lapatinib, with an 8 fold shift in IC50 towards resistance, indicating reduced expression of the protein could be involved in the resistance machinery. Conclusions: miR-127, miR-409, and miR-495, clustered to the 14q32 region are subject to epigenetic control, which is altered in lapatinib resistant cells. Resulting upregulation of the miRNAs appears to post-transcriptionally regulate genes, such as BASP1, involved in an acquired resistance mechanism to lapatinib in HER2 positive breast cancer cell lines. Citation Format: Juliette Chupin, Ai Nagano, Victoria Haley, Catherine Lenihan, Francesca Cavicchioli, Karen O’Leary, Natasha Sahgal, Cristiana Lo Nigro, Alice Shia, Claude Chelala, Peter Schmid. miRNAs in the 14q32 cluster are involved in lapatinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2017-104

Abstract 271: Influence of autophagy modulation on synergistic interactions of lapatinib and mTOR targeted agents in HER2-amplified lapatinib resistant breast cancer models in vitro and in vivo

Abstract Background: Resistance against HER2 targeted agents ultimately limits the therapeutic success in patients with HER2-positive breast cancer. It was shown that PIK3CA mutations contribute to lapatinib resistance and achieving control of a downstream PI3K/mTOR signaling is necessary for optimal effectiveness of HER2 blockade. We and others have shown that catalytic mTORC1/2 inhibitors reverse lapatinib resistance and inhibit growth of HER2-overexpressing breast cancer models in vitro and in vivo. However, activity of these targeted agents is hindered by the compensatory and adaptive mechanisms that arise to assure cell survival. One of the pro-survival responses is cytoprotective autophagy induced by lapatinib and mTORC1/2 inhibitors. Thus, we examined whether impairing autophagy could augment activity of lapatinib/mTORC1/2 inhibitors combinations in lapatinib-resistant breast cancer models. Methods: The combination of lapatinib with catalytic mTORC1/2 inhibitors KU-0063794 (KU) or AZD2014 (AZD) was evaluated in vitro and in vivo in lapatinib resistant PIK3CA mutated HER2-overexpressing/amplified MDA-MB-361, JIMT-1 and MDA-MB-453 breast cancer models in the presence of siRNA-based (Atg7, Beclin-1) and pharmacological (hydroxychloroquine (HCQ)) inhibitors of autophagy. Results: In vitro lapatinib/mTORC1/2 combinations elevated autophagy to a greater extent that either compound alone. Genetic or pharmacological inhibition of treatment-induced autophagy further decreased cell viability, suggesting that autophagy was playing a cytoprotective role in this context. In vivo, lapatinib and AZD combinations achieved effective tumor growth inhibition of 98%, 111% and 152% in MDA-MB-361, JIMT-1 and MDA-MB-453 models respectively, however addition of HCQ did not significantly enhance this therapeutic response (p>0.05). Conclusion: Negligible effects of HCQ in vivo in tumors treated with lapatinib/AZD combinations may be attributed to ineffective inhibition of autophagy-mediated survival signals that, if significantly blocked, could increase efficacy of the treatment. Utilizing carrier nanotechnology to optimize delivery of HCQ to the tumor site and molecular analysis of HCQ-engendered off target effects on survival and proliferation pathways in tumor tissue are being pursued. Citation Format: Wieslawa H. Dragowska, Sherry A. Weppler, William Wei Chu, Norman S. Chow, Jenna S. Rawji, Ashleen S. Prasad, Karen A. Gelmon, Sharon M. Gorski, Marcel B. Bally. Influence of autophagy modulation on synergistic interactions of lapatinib and mTOR targeted agents in HER2-amplified lapatinib resistant breast cancer models in vitro and in vivo. [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 271.

Abstract 319: Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance

Abstract Drug resistance is a major limitation of treatment regimens targeting the human epidermal growth factor receptor-2 (HER2) in breast cancers that overexpress this protein. Therefore, identification of novel drug targets to overcome anti-HER2 drug resistance is an unmet clinical need. Our previous studies have suggested that GPR110 may be a potential target for anti-HER2 drug resistance and tumorigenicity. Furthermore, GPR110 is an orphan receptor in the adhesion G-protein coupled receptor family, members of which have shown a role in cancer metastasis. In this study, our objectives were (1) to assess the gene expression of GPR110 using the Taqman RT-PCR assay in anti-HER2 drug resistant and in cancer stem-like cell population of various HER2+ breast cancer cell line models and (2) to investigate the effects of GPR110 gene knockdown on tumorigenesis and metastasis using various cell-based assays in HER2+ breast cancer cell lines and their anti-HER2-resistant derivatives. Cells transfected with non-targeting and 3 independent GPR110-targeting siRNAs were used to assess anchorage-independent cell growth, mammosphere formation, and cell invasion and migration. We found that GPR110 gene expression was significantly higher (>/ = 2-fold) in lapatinib-resistant (LR), trastuzumab-resistant (TR), and lapatinib + trastuzumab-resistant (LTR) derivatives of BT474, SKBR3 and UACC812 cell lines compared to the parental cells. GPR110 gene expression was also significantly higher (>/ = 1.9-fold) in ALDH+ versus ALDH- cell populations of BT474, SKBR3, MDA-MB-361 and HCC1569 cell lines. In addition, GPR110 gene knockdown led to a 55-85% (p<0.05) decrease in anchorage-independent cell growth in BT474 and SKBR3 LTR derivatives but not in parental cells. A significant reduction in mammosphere count upon GPR110 knockdown was also seen in LTR derivatives (50-70%, p<0.05) and not in parental BT474 cells. Interestingly, GPR110 knockdown in SKBR3 parental cells resulted in a significant reduction in cell invasion (40-50%, p<0.05) and migration (40-50%, p<0.05) suggesting a potential role of GPR110 in tumor cell dissemination. Overall, these results demonstrate that GPR110 is overexpressed in multiple anti-HER resistant derivatives as well as breast cancer stem-like cell population of various HER2+ breast cancer cell line models. Our studies also suggest a role of GPR110 in tumorigenesis specifically in the context of anti-HER2 drug resistance and in metastatic processes. Future in vivo studies will assess GPR110 as a novel drug target to overcome anti-HER2 drug resistance and combat metastasis in HER2+ breast cancer. Citation Format: Debashish Sahay, Raksha Bhat, Motthafar Rimawi, C. Kent Osborne, Rachel Schiff, Meghana V. Trivedi. Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance. [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 319.

Abstract B69: Acquisition and maintenance of anti-Her2 target therapies resistance in breast cancer: Role of fibrobroblasts

Abstract Introduction: Mechanisms underlying tumor progression after chemotherapy are not well understood. Therapeutic treatments can favor the clonal selection of cells with unique properties and different fitness for a given microenvironment. Indeed, tumor cells can induce changes in the structure and composition of the microenvironment to support their growth and spread. Our aim is to study if clonal selection induced by target therapies like Trastuzumab and Lapatinib favors the outgrowth of cells with different microenvironmental crosstalk capability, and in particular the role of fibroblast in the selection of these particular clones. Materials and Methods: In order to investigate if clonal selection induced by target therapies like Trastuzumab and Lapatinib favors the outgrowth of cells with different capability of microenvironmental crosstalk, we developed different cell lines resistant to these drugs from the parental SKBR3, BT474 and MDA-MB-453 Her2+ cells lines. We determined their molecular profile and investigated the changes in the expression of selected genes codifying soluble factors known to induce stromal changes, like chemokines, cytokines, matrix remodeling-related enzymes, angiogenic and neurogenic factors. To study the role of fibroblast in the selection of the resistant clones, and the crosstalk between these two populations, we developed fibroblast immortalized lines derived from breast cancer tumors and normal breast tissue, and study the effect of the fibroblasts in resistant induction, as well as the effect of resistant clones in the fibroblast activation. Results and Discussion: We observed that the major changes in the genes investigated were obtained in the Lapatinib resistant cell lines, suggesting that the acquisition of Lapatinib resistance can provide the tumor with a higher capability of stromal interaction. In vivo, the resistant cell lines showed an invasive growth pattern and higher angiogenesis compared to the parental cell lines. We observed a different pattern of fibroblast distribution in the tumors derived from the resistant cell lines, which display a more infiltrative distribution. The resistant tumors were also more fibroblast-enriched suggesting a higher microenvironment crosstalk capacity. In vitro, we found that the supernatant of breast cancer associated fibroblast was able to induce an increase in the resistant phenotype of parental lines, and that the resistant cell lines were able to induce a slightly activation of fibroblasts. Conclusion: These results highlight the importance of microenvironment in supporting tumor progression after chemotherapy. Citation Format: Patricia Fernandez-Nogueira, Mario Mancino, Gemma Fuster, Estel Enreig, Elisabeth Ametller, Paloma Bragado, Vanessa Almendro, Pere Gascon. Acquisition and maintenance of anti-Her2 target therapies resistance in breast cancer: Role of fibrobroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B69.