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Abemaciclib has activity in xenograft models of triple negative breast cancer with intact Rb-signaling

PDF file, 66K, Quantification of proteins from Western blots in Supplementary Figure S7 by densitometry using ImageJ software.

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Supplementary Figure 2 PDF file - 91K, Speed of recovery of downstream targets in NCI-N87

Whole exome sequencing analysis of a panel of 44 breast cancer cell lines, enriched for somatic alterations with functional consequence in cancer

Supplementary Table 1 PDF file - 69K, NVP-AUY922 IC50 concentrations and response to trastuzumab in HER2 amplified breast cancer cell lines

PDF file, 77K, Comparison of the effect of dacomitinib at 48 hrs and 5 days on cell cycle and apoptosis in BT-474 and SK-BR-3 lines.

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PDF file, 60K, Effect of dacomitinib on apoptosis in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 68K, Quantification of proteins from Western blots in Figure 3 by densitometry using ImageJ software.

Supplementary Legends PDF file - 78K, Supplementary Figure Legends

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2− and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2−, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2−, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897–907. ©2018 AACR.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

PDF file, 247K, The effects of dacomitinib on protein expression in lines resistant to trastuzumab and lapatinib.

Supplementary Figure 3 PDF file - 58K, Activation of HSP70 in both N87-Parental and N87-TRC cell lines

PDF file, 75K, Dacomitinib response is marginally inversely correlated with relative HER3 mRNA levels.

PDF file, 50K, Effect of dacomitinib on cell cycle in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 45K, Dacomitinib response is associated with the total HER2 protein levels in HER2-amplified cell lines.

PDF file, 60K, Dacomitinib response is associated with relative HER2 mRNA levels.

File containing three supplemental tables listing the mean tumor volumes and responses in the ER+/HER2-, HER2+/ER+ and TNBC xenografts

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PDF file - 40K, Figure S2: Biomarkers of in vivo response to inhibitors of PI3K/mTOR signaling. Reverse Phase Protein Analysis (RPPA) of KPL-1 tumor samples taken after 7 days treatment with the molecules indicated. Tumors taken 24 hours post final dose. A, pAKT Ser473. B, pS6 Ser235/236.

PDF file - 36K, Figure S3. Xenograft response data for 7-day tissue acquisition studies. A-C, SUM190, UACC812 and KPL-1 xenografts (6 mice per arm) were treated with BKM120 at 35 mg/kg, BYL719 at 50 mg/kg, RAD001 at 10 mg/kg and BEZ235 at 30 mg/kg, all daily PO. Trastuzumab was given at given at 10 mg/kg IP twice weekly. For SUM190 (A), tumors were collected 2.5 hours post final dose, for UACC812 (B) and KPL-1 (C), samples were collected 24 hours post final dose.

PDF file - 772K, Figure S1: Targeting the PI3K/mTOR pathway in HER2-amplified breast cancer cell lines. Effect of A, RAD001, B, BEZ235, C, BKM120 or D, BYL719 treatment on PI3K/mTOR signaling in two representative cell lines; SKBR3 and MDA453 (PIK3CA mutant H1047R). All lysates taken 24 hours post-treatment.

XLSX file - 213K, Table S2. Summary of in vivo activity of BKM120, BYL719, RAD001 and BEZ235 in breast cancer tumor models. T/C: Ratio of mean tumor volume of treated (T) to control (C) at final day of treatment. Final day was determined as the final day of treatment for the vehicle control arm for each study in Figure 5. Comparisons between groups were all made by Repeated Measures ANOVA (RMANOVA) followed by Dunnett test for multiple comparisons. BW: body weight; TR: treatment related; NTR: non-treatment related.

XLSX file - 14K, Table S1: Calculated IC50s for the PI3K/mTOR pathway inhibitors against the breast cancer cell line panel. ER: Estrogen Receptor. N/A: IC50 not achieved, N/D: IC50 not determined

The development of multiple PARP inhibitors in BRCA mutant cancers has been one of the most successful implementations of molecularly targeted therapies in oncology. The recent approval of niraparib in BRCA WT Ovarian cancers demonstrates the potential for expansion of PARP inhibitors to other BRCA WT indications. The successful development of PARP inhibitors into new cancer indications will depend on using molecular biomarkers to identify tumors with a unique dependency on PARP1/2 activity. We screened a large (N=523), diverse panel of human cancer cell lines for response to three approved PARP inhibitors: olaparib, niraparib, and talazoparib. Despite differences in relative potency, there was a strong correlation between sensitive and resistant cell lines to all three compounds. In vitro experimentation revealed the inhibitors share a similar biologic mechanism of response, namely a global decrease in PARylation, as well as PARP-trapping at DNA nicks leading to G2 arrest and ultimately cell death. A suite of baseline genomic and proteomic characteristics were found to be strongly associated with response to PARP1/2 inhibition, and were largely consistent between different cancer types. Special attention was paid to BRCA1/2 variants, which represent the only approved patient selection biomarkers. Notably, neither mutations in BRCA1 nor BRCA2 were found to be associated with response to PARP1/2 inhibition in our panel, even when restricting to those with confirmed deleterious ClinVar scores. The molecular predictors of response identified in our screen may ultimately be used to develop diagnostic tools for enrollment into biomarker-enriched clinical trials to expand the use of this promising class of drug into areas of high unmet need. Triple-negative breast cancer represents one of the most intriguing spaces for development. The clinical failures in this space may be partially explained by the lack of empirical predictive biomarkers in the trial design. Our diagnostics may finally allow for the efficacious deployment of PARP inhibitors to triple-negative breast cancer. In particular we have identified a panel of biomarkers that can predict for sensitivity to PARP inhibitors in breast cancer that could be used as patient selection criteria for the expanded clinical development of these compounds into PARP-naïve patient populations with high unmet need. Citation Format: Martina SJ McDermott, Dylan F Conklin, Neil A O'Brien, Kevin Chau, Dennis J Slamon. Pan-cancer analysis of PARP inhibition reveals a suite of biomarkers that correlate with PARP1/2 activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-07.

Background: Pharmacologically targeting the HER2 oncoprotein provides clinical benefit for patients with HER2-amplified cancers. However, a significant number of patients do not respond to the currently approved HER2-targeted therapies, despite carrying the HER2-alteration. Small molecule inhibitors of HER2, that target other receptor tyrosine kinases such as EGFR (i.e. lapatinib), are approved and provide some clinical benefit but are often associated with increased toxicity. Tucatinib (ARRY-380) is an orally available, potent, highly selective small molecule inhibitor of the HER2 kinase. In this study, we assessed the in vitroand in vivoactivity of tucatinib, relative to approved HER2-targeting molecules, in a panel of molecularly characterized breast cancer cell lines. Materials and Methods:The growth inhibitory activity of tucatinib, trastuzumab and lapatinib were evaluated in a panel of 48 breast cancer cell lines molecularly characterized at baselineby genomic (array-CGH) and proteomic (Reverse Phase Protein Array; RPPA) profiling. IC50values for tucatinib and lapatinibwere determined from direct cell counts using a Cellavista Cell Imaging System. Trastuzumab activity was measured as % inhibition of cell growth at fixed concentrations. In vivoefficacy of tucatinib was assessed in cell line xenograft models of HER2+/ER- and HER2+/ER+ breast cancers as a single agent or in combination with targeted therapies for breast cancer. Results: A broad range of IC50values (3.2nM to >10μM), was seen for tucatinib with a high degree of selectivity for the HER2-amplfied sub-type. High levels of total and phosphorylated HER2 (pHER2) accompanied by high levels of pEGFR and pHER3 enriched for sensitivity to tucatinib, confirming that HER2-driven cancers may be uniquely sensitive to tucatinib. The response profile for lapatinib was less clean, with responses also observed in HER2-low/EGFR-high cell lines. Sensitivity to tucatinib was also observed in HER2-amplified cell lines that were either de novoor acquired resistant to trastuzumab. Single agent tucatinib induced tumor regressions in a xenograft model of HER2+/ER+ breast cancer. Tumor regressions were further enhanced by combination with trastuzumab. The combination of tucatinib plus trastuzumab was as efficacious and better tolerated than trastuzumab plus docetaxel or trastuzumab plus pertuzumab plus docetaxel. The triple combination of tucatinib plus hormonal blockade (fulvestrant) and CDK4/6 inhibition (abemaciclib) also induced robust tumor regressions, without significant body weight loss. Discussion: These preclinical data highlight the potential of the HER2-selective small molecule inhibitor, tucatinib, to provide benefit to patients with HER2-amplifed cancers. Furthermore, our biomarker analysis of response to tucatinib has identified a HER2-driven signature within the HER2-amplfied sub-type that selects for sensitivity to tucatinib. Selecting patients based on this profile may further enrich for individuals most likely to benefit from tucatinib-based therapies. Citation Format: O'Brien NA, Conklin D, McDermott M, Luo T, Ayala R, Issakhanian S, Salgar S, Hurvitz S, Slamon DJ. The small molecule inhibitor of HER2, tucatinib, has potent and highly selective activity in preclinical modes of HER2-driven 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 P6-17-11.

Background: Claudin 6 (CLDN6), a member of the claudin family of tight junction proteins, is expressed at high levels in multiple human malignancies including ovarian and endometrial cancers. Conversely it has little or no expression in normal tissues. This expression profile makes CLDN6 an ideal target for development of potential therapeutic antibody-drug conjugates (ADCs). This study describes the generation and preclinical characterization of an anti-CLDN6 ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to MMAE via a cleavable linker. Materials and Methods: A fully humanized anti-CLDN6 antibody was initially characterized for binding affinity, selectivity/specificity, internalization characteristics and in vivo efficacy. It was then conjugated to MMAE resulting in the potential therapeutic anti-CLDN6 ADC. The anti-tumor efficacy of the ADC was next assessed for anti-tumor efficacy in CLDN6 positive (CLDN6+) and negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of specific cancers including ovarian and endometrial cancer. Results: Selective binding of the ADC to CLDN6, without cross reactivity to other CLDN family members CLDN3, CLDN4 and CLDN9, was confirmed in human cancer cell lines and cells engineered to overexpress each protein. The ADC was also shown to rapidly internalize in CLDN6+ cells. Robust tumor regressions following treatment with the ADC were observed in CLDN6+ xenografts that were sustained beyond the treatment window. Conversely, there was limited to no activity of the ADC in CLDN6- xenografts models. In addition, the prevalence of CLDN6 expression in human ovarian and endometrial cancers was assessed by IHC in tissue microarrays and found to be 28% (ovarian epithelial carcinomas) and 11% (endometrial carcinomas), respectively. Discussion: Overall, these data suggest that our anti-CLDN6 ADC may be a promising treatment for patients with CLDN6+ tumors and it is currently in Phase I clinical testing. Citation Format: Martina S. McDermott, Ke Wei Gong, Neil A. O'Brien, Dylan Conklin, Benjamin Hoffstrom, Ming Lu, Jun Zhang, Tong Luo, Weiping Jia, Jenny J. Hong, Kevin Chau, Simon Davenport, Michael F. Press, Abram Handly-Santana, Joan S. Brugge, Ronny Drapkin, John A. Glaspy, Leonard Presta, Dennis J. Slamon. Development and characterization of a novel anti-CLDN6 antibody drug conjugate for the treatment of CLDN6 positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 342.

Background: Claudin 18 is a member of the claudin family of tight junction tetraspan cell surface proteins. The 18.2 isoform of claudin 18 (CLDN18.2) is expressed in healthy gastric mucosa cells and largely absent from other normal adult cell types. In contrast to the low expression in healthy tissues, CLDN18.2 is highly overexpressed in many cancer types including gastric and pancreatic malignancies at high frequencies (50-80%). The high tumor-normal expression differential of CLDN18.2 make it an attractive target for both therapeutic monoclonal antibodies (mAbs) and antibody drug conjugate (ADCs). Here, we describe the preclinical development of a novel therapeutic CLDN18.2 mAb and ADC. Materials and Methods: CLDN18.2 specific mAbs were generated through a multiplexed immunization strategy by which mice were immunized with either peptides spanning loop 2 of the CLDN18.2 extracellular domain or mouse NIH3T3 cells overexpressing the full length CLDN18.2 protein. Antibody clones were screened by flow cytometry for selective binding to cell surface CLDN18.2. A CLDN18.2-directed ADC was generated from the fully humanized clinical candidate CLDN18.2 mAb by MMAE conjugation with a cleavable linker. CLDN18.2 positivity in cell line (CDX) and patient derived xenograft (PDX) models was determined by IHC assay. Results: Selective binding of the CLDN18.2-mAb to CLDN18.2, without cross reactivity to the more widely expressed CLDN18.1 isoform, was confirmed in human cancer cell lines and cells engineered to overexpress each isoform. The CLDN18.2-mAb demonstrated strong induction of both antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in in vitro assays; suggesting that Fc effector function is a key component of the mechanism of action of the mAb. Treatment of CLDN18.2 positive gastric and pancreatic CDXs with CLDN18.2-mAb monotherapy induced significant inhibition of tumor growth in CD-1 nude mice. Binding of CLDN18.2-mAb or CLDN18.2-ADC to the cell surface CLDN18.2 induced internalization of the protein-antibody complex. Complete regression of CDX tumors was observed in response to treatment with the CLDN18.2-ADC in three separate CLDN18.2 positive models (two pancreas, one gastric). Moreover, complete loss of xenograft tumor burden was also observed in 4/5 CLDN18.2 positive pancreas PDX models. No impact on xenograft tumor growth as observed in response to treatment with either the CLDN18.2-mAb or CLDN18.2-ADC in CLDN18.2 negative CDX/PDX models. Discussion: These data support the clinical development of this CLDN18.2-mAb and CLDN18.2-ADC for the treatment of CLDN18.2 positive cancers. Each of these molecules will begin phase 1 clinical testing in early 2022. Citation Format: Neil A. O'brien, Martina S. McDermott, Jun Zhang, Ke Wei Gong, Ming Lu, Benjamin G. Hoffstrom, Dylan Conklin, Tong Luo, Kevin Chau, Min Liang, Timothy R. Donahue, John A. Glaspy, Leonard Presta, Dennis J. Slamon. Development of a novel therapeutic CLDN18.2 monoclonal antibody and antibody drug conjugate for the treatment of CLDN18.2 positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 332.

Journey to jorumycin Jorumycin is a structurally complex, pentacyclic organic compound produced by a marine mollusk. The success of a similar compound, trabectedin, in treating certain types of cancer has focused attention on exploring jorumycin's pharmaceutical properties. Welin et al. developed a succinct route to synthesizing jorumycin and the closely related jorunnamycin A that deliberately diverges from the putative biosynthetic pathway underlying prior chemical syntheses. This route, which hinges on a carefully optimized asymmetric catalytic hydrogenation, can be easily modified to introduce unnatural structural diversity for functional optimization in further drug discovery research. Science , this issue p. 270

SUMMARY To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer., In Brief The overall survival of patients with late-stage ovarian cancer is dismal. To identify therapeutic opportunities, Papp et al. integrate genomic, epigenomic, and expression analyses to provide a resource of molecular abnormalities in ovarian cancer cell lines and use these to identify tumors sensitive to PARP, MEK, and PI3K inhibitors., Graphical Abstract

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2− and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2−, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2−, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897–907. ©2018 AACR.

Poly (ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock-down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock-down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock-down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation.

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Addition of CDK4/6 inhibitors such as palbociclib, ribociclib or abemaciclib to endocrine-based therapies significantly improves progression-free and in some subgroups, overall survival in patients with advanced estrogen receptor-positive (ER+) breast cancer. However, acquired resistance to CDK4/6 inhibitors remains a significant unmet clinical need. It is critical to ascertain the mechanisms by which tumor cells evade CDK4/6 based therapy. In this study we screen multiple in vitro and in vivo models of acquired resistance to CDK4/6 inhibitors to identify potential resistance/escape pathways and targets for pharmaceutical intervention to overcome resistance. ER+ breast cancer cell lines of diverse molecular backgrounds were conditioned to acquire resistance to CDK4/6 inhibitors through either long-term culture in the presence of clinically relevant concentrations of inhibitors or as cell line xenografts treated through progression on a CDK4/6 inhibitor plus fulvestrant. Baseline and pharmacodynamic changes in cell signaling were measured using reverse phase protein array (RPPA) and RNAseq analysis. Acquired resistance to CDK4/6 inhibitors was associated with decreases in phosphorylated-Rb (pRb) and ER-alpha protein and increases in pAKT and pS6 relative to isogenic controls. The p110α-selective PI3K-inhibitor, alpelisib, in combination with fulvestrant or ribociclib/fulvestrant blocked pAKT signaling in xenografts progressing on palbociclib/fulvestrant and induced significant tumor regressions. Apelisib plus fulvestrant also produced robust anti-tumor responses in xenografts progressing on ribociclib/fulvestrant. Triple combination treatment with ribociclib/alpelisib/fulvestrant induced significant regressions in resistant tumors and in treatment naïve models, where complete tumor regressions occurred regardless of PIK3CA mutation status. Regressions were maintained for >9 weeks post withdrawal of treatment, indicating that therapeutic resistance may be prevented by this triple combination approach. RPPA analysis of responding tumors identified sustained inhibition of both PI3K- and CDK4/6:Rb-pathway signaling accompanied by activation of pro-apoptotic proteins. These data support clinical investigation of targeting PI3K with alpelisib in breast cancers progressing on CDK4/6 based therapies and investigation of upfront triple combination therapy prior to acquisition of resistance to CDK4/6. Citation Format: Neil A. O'Brien, Martina SJ McDermott, Dylan F. Conklin, Alex Gaither, Tong Luo, Raul Ayala, Suruchi Salgar, Emmanuelle DiTomaso, Naveen Babbar, Faye Su, Sara A. Hurvitz, Ronald Linnartz, Kristine Rose, Samit Hirawat, Dennis J. Slamon. Targeting activated PI3K/mTOR signaling overcomes resistance to CDK4/6-based therapies in preclinical ER+ breast cancer models [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 3825.

Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines. Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis. Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment. Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML. Citation Format: Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML) [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 297.

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p = 0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p = 0.004) and low baseline p21 protein levels (RR = 2.28, p = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (

Poly (ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock-down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock-down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock-down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation.

Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA, and drug-screening data have not been available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic, and drug-screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

Background: Copanlisib (BAY 80-6946) is a reversible, pan-class I PI3K inhibitor with preferential activity for the alpha isoform, which may be of particular importance in multiple myeloma. Here we demonstrated the in vitro efficacy of copanlisib in a panel of 20 multiple myeloma cell lines. Baseline levels of phospho-S6 (P-S6) correlated with sensitivity to copanlisib, resulting in a potential biomarker of response. In addition, the change of P-S6 post-treatment could be used as a pharmacodynamic biomarker for copanlisib treatment. Methods: We screened a panel of 20 multiple myeloma cell lines and selected 3 sensitive: NCI-H929, MM.1S, L-363, and 3 resistant: AMO-1, JJN3, COLO-677 for further analysis. We performed apoptosis and cell senescence assays following 72 hours of 50nM and 100nM copanlisib exposure. Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or PI/ANX-V FITC staining, respectively. Cellular senescence was determined by measuring β-galactosidase activity in cells treated for 96 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Flow cytometry was also performed to monitor the post-treatment P-S6 level changes. Results: Copanlisib treatment induced apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). An increased cell cycle arrest in G1 was also observed in the sensitive cell lines but not in the resistant lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. RPPA analysis demonstrated lower baseline p-S6 (S235/236, S240/244) protein levels in sensitive compared to resistant cell lines and this was confirmed with western blot analysis. Treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) than in the resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively), which was validated by western blot and phospho-flow. We also showed by RPPA and WB that copanlisib down-regulates pro-survival and proliferation molecules including p-S6K1, p-S6 and p-4EBP1, and upregulates pro-apoptotic PDCD4 in all cell lines, but to a greater extent in sensitive cell lines. Finally, pharmacodynamic p-S6 response remained at different post-treatment time points. Discussion: A differential response to copanlisib is seen in the myeloma cell line panel. A subgroup of multiple myeloma cell lines demonstrated median IC50 values in the low nanomolar range (5-100nM), and responses correlated with low baseline P-S6. This p-S6 stratified response was only observed with PI3K-alpha inhibitors, but not with inhibitors targeting other PI3K isoforms or pan-PI3K inhibitors. Further studies may allow development of a new patient screening method or companion diagnostic. Citation Format: Sarah Larson, Mao Yu Peng, Monica Mead, Andrae Vandross, Dylan Conklin, Erika Von Euw, Dennis J. Slamon. Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma [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 136. doi:10.1158/1538-7445.AM2017-136

The selective cyclin dependent kinases 4 and 6 (CDK-4/6) inhibitor, palbociclib, has recently been approved in combination with endocrine-based therapies for the treatment of advanced ER+/HER2- breast cancer (BC). Despite the improvements in progression-free and overall survivals, a significant number of patients on CDK-4/6 therapy will go on to develop progressive disease. As such, it is critical to identify the mechanisms by which tumor cells evade CDK-4/6 targeted therapy and to develop therapeutic strategies to overcome resistance. In this study, we evaluated the efficacy of a p110α-selective PI3K inhibitor, alpelisib (BYL719) and the mTORC1 specific inhibitor, everolimus (RAD001), in ER+/HER2- xenograft tumors conditioned in vitro and in vivo to acquire resistance to combined palbociclib + endocrine-based therapy. The EFM19 (ER+/HER2-, PIK3CA mt), MDA134 (ER+/HER2-, PIK3CA wt), and MDA361 (ER+/HER2+, PIK3CA mt) BC cell lines were conditioned to acquire palbociclib resistance through long-term culture in the presence of increasing concentrations of drug. Baseline total/phosphoprotein levels of over 280 cancer-associated analytes were measured in the parental and resistant cell lines by Reverse Phase Protein Array (RPPA). In vivo resistant models were developed from xenografts of the MCF7 and HCC1500 ER+ BC cell lines, treated until progression with palbociclib (75 mg/kg) + fulvestrant (5 mg/mse). EFM19-PR cells maintained palbociclib resistance in vivo and were included in the xenograft studies. Resistance to palbociclib was confirmed in vitro by a shift in growth inhibitory IC50 from an average of less 50nM to over 1 µM. Significantly reduced Rb and ER protein levels were detected in each of these palbociclib resistant cell lines by RPPA. Cross-resistance to an alternative CDK-4/6 inhibitor (ribociclib) was found in in vitro studies. In addition, switching treatment of the xenografts progressing on palbociclib + fulvestrant to ribociclib (75mg/kg) + fulvestrant did not impact tumor progression. However, when progressing tumors were switched to BYL719 (30mg/kg) + fulvestrant or RAD001 (10mg/kg) + fulvestrant, sustained tumor regression was observed for over 45 days of treatment in every model tested. The triplet combination of ribociclib + fulvestrant + BYL719 or RAD001 provided marginal additional benefit over the doublet combination of BYL719 or RAD001 + fulvestrant. RPPA analysis of xenograft tissue collected from these studies will help to identify additional molecular alterations involved in resistance to CDK-4/6 targeted therapy. These preclinical data indicate that acquired resistance to CDK-4/6 inhibition occurs through a loss of dependence on Rb-signaling. However, targeting an alternative pathway like PI3K/mTOR with molecules such as BYL719 or RAD001 may be a viable strategy for further clinical investigation. Citation Format: Neil A. O'Brien, Dylan Conklin, Tong Luo, Raul Ayala, Shawnt Issakhanian, Ondrej Kalous, Erika Von Euw, Sara A. Hurvitz, Emmanuelle DiTomaso, Faye Su, Ronald Linnartz, Stefan Scherer, Samit Hirawat, Dennis J. Slamon. Anti-tumor activity of the PI3K/mTOR pathway inhibitors alpelisib (BYL719) and everolimus (RAD001) in xenograft models of acquired resistance to CDK-4/6 targeted therapy [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 4150. doi:10.1158/1538-7445.AM2017-4150

Purpose: Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies. Experimental Design: The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110α isoform–specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines. Results: Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor–positive and/or HER2+ cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2+ breast cancer models. The PI3K/mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1–2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo. Conclusion: These preclinical data support the findings of the BOLERO 3 trial that shows that targeting of the PI3K/mTOR pathway in combination with trastuzumab is beneficial in trastuzumab-resistant breast cancer. Clin Cancer Res; 20(13); 3507–20. ©2014 AACR.

PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines.Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression.IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041).PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.