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Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis that accounts for 10-20% of breast cancer cases worldwide. Intra-tumoral heterogeneity and tumor cell plasticity are thought to contribute to drug resistance in TNBCs. Our work aims to: 1) precisely identify the intra-tumoral heterogeneity in cellular states present within TNBC, and 2) test whether drugs can block or initiate plasticity between subpopulations to improve drug sensitivity. We hypothesize that treatment(s) induce cellular plasticity, thus causing cells to shift into resistant states that persist until treatment is removed. We further hypothesize that these resistant subpopulations give rise to new tumor outgrowths once the treatment stops. Methods: To test this, we are treating TP53-/- Genetically Engineered Mouse Model (GEMM) syngeneic transplant tumors of the basal-like TNBC phenotype with the chemotherapeutic doublet of carboplatin/paclitaxel, and targeted agents implicated in plasticity including the MEK inhibitor trametinib, a chromatin remodeling inhibitor I-BET151, and the dihydroorotate dehydrogenase inhibitor brequinar. To identify cellular subpopulations and examine their response to treatment, we performed both in vivo and in vitro drug sensitivity testing, as well as gene expression profiling using single cell RNA-sequencing (scRNAseq). Results: We have identified clear intra-tumoral heterogeneity with at least 6 distinct cell states present, including basal, mesenchymal/claudin-low, and proliferative subpopulations in vivo in most TNBC GEMM models. We performed 18 individual scRNAseq experiments on the TP53-/- 2225L GEMM transplant line with the aforementioned treatments and untreated controls in triplicate and compared subpopulation frequencies in treated versus untreated tumors. Notably, treatment with trametinib and brequinar caused the rise of two rare subpopulations (i.e. 1% to 3-4% of total tumor cells) that express genes consistent with previously described drug-tolerant persisters (DTP), which we have called “Epithelial-DTP” (Tacstd2, Krt6a, and Cryab enriched) and “Mesenchymal-DTP” (Snai2 and Sca-1 enriched). A gene signature generated from the Epithelial-DTP subpopulation predicted poor patient outcomes in neoadjuvant chemotherapy treated TNBC patients. Further, in TNBC patient-derived xenografts (PDX), these two DTP subpopulations are also present and induced by treatment to an even greater frequency. Ongoing experiments include the use of fluorescence-activated cell sorting to isolate and functionally test the tumor-initiating capabilities of these two rare cell subpopulations. In addition, many experiments are underway to identify means to therapeutically target these DTP cells, with these results to be presented. Ultimately, identifying these rare drug resistant subpopulations, and identifying means to eradicate them, could vastly improve therapeutic regimens and outcomes for patients with TNBCs. Citation Format: Cherise R. Glodowski, Kevin R. Mott, Denis Okumu, Michael P. East, Timothy C. Elston, Gary L. Johnson, Charles M. Perou. PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-09.

Since the US Food and Drug Administration (FDA) began monitoring the quality of pharmaceutical manufacturing by enforcing current good manufacturing practices roughly 60 years ago, forces related to the global economy have changed, rendering the task of monitoring quality more difficult. Alternative strategies by groups like Valisure, LLC, and the University of Kentucky Drug Quality Study to monitor the quality of the currently circulated US drug supply through end-product testing and screening have resulted in several concerning findings. Given the successful approaches of identifying quality defects in pharmaceuticals by non-regulatory bodies, and considering the changing landscape and pressures on manufacturing, the FDA, large buying groups, and the US Department of Defense should consider these alternative strategies as a means to augment current regulatory activities.

PDF - 164KB, Jurkat and 697 cell cultures were treated with UNC569 (500 nM) for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Mer. Cells were harvested after 24 and after 48 hours. Mer was immunoprecipitated from cell lysates and total Mer protein and Mer phosphoprotein (p-Mer) were detected by western blot.

File includes drug synergy scores for 1 cell line screened against OTX015

WES Coding Mutations

Supplementary Figure 2

Supplementary Table 2

MIB/MS data

File contains information on small molecules included in screening library

File includes AlphaScreening values for activity of the screening library against the BET or p300 bromodomains

Supplementary Methods

Drug Resistant POS Cells Emerge Following Chronic Trametinib Treatment.

PDF - 172KB, Jurkat cell cultures were treated with the indicated concentrations of UNC569 for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Tyro3. Tyro3 was immunoprecipitated from cell lysates and total Tyro3 protein and Tyro3 phosphoprotein (p-Tyro3) were detected by western blot. Numbers on the right indicate the location of molecular weight (kD) markers. Immunoblots are representative of three independent experiments.

PDF - 294KB, TAM receptor expression in Jurkat cell line was detected by western blot analysis. Whole cell lysates were prepared and phosphorylated (denoted by p-) and total proteins were detected. Western blots representative of three independent experiments are shown. Blots were stripped and reprobed with anti-tubulin antibody to confirm similar protein loading.

PDF - 634KB, Eighteen fish (Myc1-Myc18) were treated with UNC569 for 2 weeks. Animals were imaged pre- and post-treatment, with GFP intensities quantified as described in the text. Ten of 18 fish (55.6%; Myc1-Myc8, Myc10, Myc16) showed >50% regressions. Six fish (33.3%; Myc9, Myc11-15) had 25-50% responses, and 2/18 (11.1%; Myc17, Myc18) progressed on treatment. Average diminution in tumor burden across the entire cohort was 47.8%. Four images are shown for each animal: Day 0 columns shows the raw image and GFP intensity plot for each fish prior to UNC treatment. Day 14 columns show post-treatment images and intensity plots. GFP Score day 14 columns show final responses, depicted as % of original cancer remaining.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein–coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy.Implications:We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Supplemental figures, figure legends, table legends, methods, and references

Supplementary Materials and Methods

Loss of the tumor suppressor NF1 leads to activation of RAS effector pathways, which are therapeutically targeted by inhibition of mTOR (mTORi) or MEK (MEKi). However, therapeutic inhibition of RAS effectors leads to the development of drug resistance and ultimately disease progression. To investigate molecular signatures in the context of NF1 loss and subsequent acquired drug resistance, we analyzed the exomes, transcriptomes, and kinomes of Nf1-mutant mouse tumor cell lines and derivatives of these lines that acquired resistance to either MEKi or mTORi. Biochemical comparisons of this unique panel of tumor cells, all of which arose in Nf1+/− mice, indicate that loss of heterozygosity of Nf1 as an initial genetic event does not confer a common biochemical signature or response to kinase inhibition. Although acquired drug resistance by Nf1-mutant tumor cells was accompanied by altered kinomes and irreversibly altered transcriptomes, functionally in multiple Nf1-mutant tumor cell lines, MEKi resistance was a stable phenotype, in contrast to mTORi resistance, which was reversible. Collectively, these findings demonstrate that Nf1-mutant tumors represent a heterogeneous group biochemically and undergo broader remodeling of kinome activity and gene expression in response to targeted kinase inhibition.

File includes drug synergy scores for 4 cell lines screened against CPI-637

File includes drug synergy scores for 6 cell lines screened against JQ1

File contains enrichment scores and p-values for gene sets evaluated using GSEA for RNA sequencing data sets

Supplementary Table 1

PDF - 358KB, Cells were plated in methylcellulose in the presence of the indicated concentrations of UNC1653 or vehicle only. Colonies were counted after eight days. Long-term colony growth of Jurkat cells in methylcellulose is shown. Mean values and standard errors were derived from 3 independent experiments.

Supplemental Figure Legends

Abemeciclib competitive MIB/MS TMT dose response

Expanded detail on MIB MS experiment

Structural reorganization and amelanotic intratumoral nests are evident in persistent melanoma on MEKi.

PDF file, 53K, MP1U Dosing Regimens.

Kinome and transcriptome responses to combined BRAF and MEK inhibition in murine tumors, patient samples, and human cell lines

Pharmacodynamic changes (pathways)

Response markers

Extended Materials and Methods with in depth details about experimental procedures and protocols

Graphical representation and model of results

Pharmacodynamic markers

mRNAseq pathway signature analysis reveals gene expression reprogramming on MEKi.

mRNA isoforms

MEKi Experiment Overview

BRAFV600E and PTEN-loss are required for tumor growth, which is temporally controlled and reproducible at the cell level.

Supplementary tables 1 and 2; Clinical information about the patients that the PDX models were established from and pathways that are regulated in the sensitive tumors after buparlisib treatment, respectively.

Purpose: Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.Experimental Design: We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).Results: Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein–extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the “AZD/BEZ” combination in the melanoma GEMM, we next tested this regimen in the “claudin-low” breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2+ tumors.Conclusion: These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies. Clin Cancer Res; 18(19); 5290–303. ©2012 AACR.

Response markers (pathways)

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

Sup Fig Legends

Achievement of reproducible spatial and temporal control of melanoma growth with

Supplementary Figures 1-10 supporting the study Mundt et al., "Proteomic profiling of PI3K inhibitor resistance"

Introduction: Translational research is essential to the success of every cancer center. Increasingly doctors ask patients to donate tissue to study all aspects of the disease to improve cancer treatments and quality of life. Patients are key stakeholders and partners in the success of the translational research process. The University of North Carolina (UNC), Lineberger Comprehensive Cancer Center (LCCC) researchers would like to increase the number of patients that donate tissue to support research and value patients who donate. Securing the right tissues at the right time, and processing and storing them properly is essential for research that leads to breakthrough discoveries. Purpose: LCCC supports the inclusion of patient advocates in all cancer center research. Therefore, the UNC Breast SPORE patient advocates initiated a program called Patients and Researchers Together (PART) to ensure patients work closely with researchers to drive the best research forward. The PART-Tissue program aims to develop a process to increase the donation and usage of patient donated tissues. This program is built on significant patient engagement that leads to a bi-directional patient and researcher PARTnership. We believe patients will be more willing to donate tissue if they are informed about the research process, understand the impact research has to improve patient’s lives, and are valued as PARTners of LCCC. Objectives: The first objective is to promote the excellence of LCCC translational research to all stakeholders, including patients and the community. Second; create a patient-centered tissue donation program that will meet researcher needs across LCCC, and Third; ensure the tissue collection process is primarily focused on recognizing patient’s voluntary contributions while minimizing patient burden. Methods: We first created a multi-stakeholder committee to ensure the participation of key stakeholders in all activities. We developed a process to identify relevant materials to assist in tissue donation at LCCC. The project focused on a Lineberger breast cancer tissue collection protocol (LCCC9819). We discussed patient, researcher and clinician/surgeon needs that reflect the vision of the PART program. We identified the need for several types of informational materials for patients, developed them with multi-stakeholder involvement and evaluated their accuracy, readability, understandability, and health literacy. We assessed the current and future needs of LCCC researchers by conducting interviews and surveys. We also engaged patient advocates as PARTners throughout this project. Results: We initiated the PART-Tissue program in August 2020 and we have engaged multiple stakeholders throughout the project. We evaluated LCCC9819 from the patient, research and clinical perspectives. We identified a series of informational materials needed for patients to introduce them to the value of tissue donation and to introduce them to the LCCC9819 protocol. We developed two introductory materials and a study summary. We conducted interviews and developed a survey to collect information about researcher needs at LCCC. In pilot interviews, we noticed a high need for fresh tissue, which requires real-time coordination between clinicians, clinical research coordinators, surgeons, pathologists and researchers. We also developed a website to connect directly with patients and the community about the importance of tissue donation that can lead to discoveries in cancer risk reduction and treatment. The goal is to develop a process where patients feel encouraged to donate tissue samples and are appreciated as partners with researchers who use the tissue to improve the lives of cancer patients. Citation Format: Patricia A. Spears, Vernal Branch, Jennifer A. Potter, Missy Van Lokeren, Nasrin H. Babadi, Adriana S. Beltran, Laurie Betts, Coleen Crespo, Barbara Dean, Amy L. Garrett, Laura T. Jensen, Gary L. Johnson, Hayley Morris, Philip Spanheimer, Charles M. Perou, Lisa A. Carey, H. Shelton Earp. Patients and Researchers Together (PART); a patient-centered tumor tissue collection PARTnership between patients and researchers to increase tissue donations for breast cancer research [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-15-01.