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PDF file 62K, MCL1 overexpression enhances docetaxel resistance in non-amplified TNBC cell lines

PDF file 77K, Association of gene alterations with gene expression

XLSX file 706K, This file contains 8 supplementary data tables which include patient demographics, genes and alterations assessed by NGS, Nanostring gene expression data, and bioinformatics analyses

Fig S1. Frequent focal amplification of DNA proximal to MAP3K8 breakpoint from TCGA tumors and overall survival. Fig S2. Breakpoint spanning reads and resulting protein translation from additional TCGA melanoma tumors with MAP3K8 rearrangements. Fig S3. Exogenous expression of full length and truncated MAP3K8 decrease sensitivity of BRAF-mutated cells. Fig S4. Identification of additional potential cell line models with MAP3K8 truncating rearrangements. Fig S5. Viability selectively decreased upon MAP3K8 siRNA knockdown in cells harboring truncating MAP3K8 rearrangements. Fig S6. Differential gene expression analysis identifies pathways enriched in MAP3K8-truncated melanomas. Fig S7. Break-apart FISH scoring of melanoma tissue microarray identifies rearranged and amplified MAP3K8.

Supplemental Figure Legends 1-7

Supplemental Table 1. MAP3K8 qPCR positional primers Supplemental Table 2. TCGA SKCM dataset clinical and expression data. Supplemental Table 3. Rockland Wistar Institution Melanoma Cell Lines Mutation Staus Supplemental Table 4. MAP3K8 break-apart FISH scoring of Vanderbilt TMA

PDF file 45K, Association of high Ki67 with basal-like status

PDF file 26K, Association of node status with RFS and OS, stratified by menopausal status

PDF 118K, TSC1 truncation in the RD of a TNBC after NAC

PDF file 1474K, MEK activity is required for MYC-induced soft agar colony formation

PDF file 64K, Legends for all supplementary data figures (1-7)

Background: Immune checkpoint inhibitors (ICI) have improved patient overall and progression-free survival in some cancer types but yielded limited success in breast cancer. Phase-III clinical trials in triple negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes within tumor stroma, have demonstrated increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Consequently, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC patients, and potentially in the early breast cancer setting. Despite FDA-approval, the therapeutic benefit of ICI alone and the most efficacious chemotherapy combinations are poorly characterized. Objective: We sought to model ICI response in vivo to elucidate the mechanisms responsible for immunotherapy efficacy in breast cancer and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI. Methods: In this study, we used an immunocompetent EMT6 orthotopic mammary tumor model to investigate the efficacy of single-agent immunotherapy and in combination with standard-of-care chemotherapy (paclitaxel [PAC] or doxorubicin [DOX]). We used single-cell RNA sequencing to analyze the cellular landscape of the primary tumor in response to combinatorial therapeutic strategies. Additionally, we serially sampled and analyzed peripheral blood from mice with differential responses by bulk and T-cell receptor (TCR) sequencing to identify systemic genetic alterations and T-cell expansion. Results: Single-agent anti-PD-L1 robustly suppressed primary tumor growth (p =0.0046) and extended survival (p Citation Format: Ann Hanna, Xiaopeng Sun, Paula I. Gonzalez-Ericsson, Violeta M. Sanchez, Melinda E. Sanders, Justin M. Balko. Host myeloid response to tumor and immunotherapy is associated with heterogeneity in outcomes to anti-PDL1 [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 P1-04-03.

Everolimus and Cisplatin Modulation of p73 and p63 Expression In Vivo.

PIK3CA mutations are associated with resistance to HER2-targeted therapies. We previously showed that HER2+/PIK3CAH1047R transgenic mammary tumors are resistant to the HER2 antibodies trastuzumab and pertuzumab but respond to PI3K inhibitor buparlisib (TPB). In this study, we identified mechanisms of resistance to combined inhibition of HER2 and PI3K. TPB-resistant tumors were generated by treating HER2+/PIK3CAH1047R tumor-bearing mice long term with the drug combination. RNA sequencing of TPB-resistant tumors revealed that extracellular matrix and cell adhesion genes, including collagen II (Col2a1), were markedly upregulated, accompanied by activation of integrin β1/Src. Cells derived from drug-resistant tumors were sensitive to TBP when grown in vitro, but exhibited resistance when plated on collagen or when reintroduced into mice. Drug resistance was partially reversed by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoate. Inhibition of integrin β1/Src blocked collagen-induced resistance to TPB and inhibited growth of drug-resistant tumors. High collagen II expression was associated with significantly lower clinical response to neoadjuvant anti-HER2 therapy in HER2+ breast cancer patients. Overall, these data suggest that upregulation of collagen/integrin/Src signaling contributes to resistance to combinatorial HER2 and PI3K inhibition. Cancer Res; 77(12); 3280–92. ©2017 AACR.

Supplementary Figure 1: Representative images of high and low sTILs.

Background: Heritable risk for breast cancer includes an increasing number of common, low effect risk variants. We conducted a multistage genetic association study in a series of independent epidemiologic breast cancer study populations to identify novel breast cancer risk variants.Methods: We tested 1,162 SNPs of greatest nominal significance from stage I of the Cancer Genetic Markers of Susceptibility breast cancer study (CGEMS; 1,145 cases, 1,142 controls) for evidence of replicated association with breast cancer in the Nashville Breast Cohort (NBC; 599 cases, 1,161 controls), the Collaborative Breast Cancer Study (CBCS; 1,552 cases, 1,185 controls), and BioVU Breast Cancer Study (BioVU; 1,172 cases, 1,172 controls).Results: Among these SNPs, a series of validated breast cancer risk variants yielded expected associations in the study populations. In addition, we observed two previously unreported loci that were significantly associated with breast cancer risk in the CGEMS, NBC, and CBCS study populations and had a consistent, although not statistically significant, risk effect in the BioVU study population. These were rs1626678 at 10q25.3 near ENO4 and KIAA1598 (meta-analysis age-adjusted OR = 1.13 [1.07–1.20], P = 5.6 × 10−5), and rs8046508 at 16q23.1 in the eighth intron of WWOX (meta-analysis age-adjusted OR = 1.20 [1.10–1.31], P = 3.5 × 10−5).Conclusions: Our data supports the association of two novel loci, at 10q25.3 and 16q23.1, with risk of breast cancer.Impact: The expanding compendium of known breast cancer genetic risk variants holds increasing power for clinical risk prediction models of breast cancer, improving upon the Gail model. Cancer Epidemiol Biomarkers Prev; 21(9); 1565–73. ©2012 AACR.

Supplementary Figure S1. RNAi screen to identify kinases that sensitize cells to CDK4/6 inhibition. Supplementary Figure S2. Calculation of the sensitivity index (SI) value. Supplementary Figure S3. PDK1 and CDK4/6 inhibitors are synergistic against a panel of cancer cell lines. Supplementary Figure S4. Inhibition of the PI3K/PDK1 pathway results in durable responses and is well-tolerated in vivo. Supplementary Figure S5. Ribociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and upregulate P-PDK1. Supplementary Figure S6. Inhibition of PI3K/PDK1/AKT/mTOR signaling pathway resensitizes ribociclib-resistant cells to CDK4/6 inhibition.

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Supplementary Tables - PDF file 148K, Table S1. Characteristics of HER2-negative breast cancers, separated by HR status, TCGA Table S2. Characteristics of triple-negative breast cancers with residual disease post neoadjuvant chemotherapy and univariate Table S3. Bimodal analysis of metabolism-related genes ranked by bimodality index (BI)

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Everolimus Inhibited mTOR Activity as Assessed by pS6 Expression without Significant Correlation with Clinical Activity

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor–positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K–PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488–99. ©2017 AACR.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

PDF file, 36K, Number of risk allele carriers.

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Purpose: Because of inherent disease heterogeneity, targeted therapies have eluded triple-negative breast cancer (TNBC), and biomarkers predictive of treatment response have not yet been identified. This study was designed to determine whether the mTOR inhibitor everolimus with cisplatin and paclitaxel would provide synergistic antitumor effects in TNBC.Methods: Patients with stage II/III TNBC were enrolled in a randomized phase II trial of preoperative weekly cisplatin, paclitaxel and daily everolimus or placebo for 12 weeks, until definitive surgery. Tumor specimens were obtained at baseline, cycle 1, and surgery. Primary endpoint was pathologic complete response (pCR); secondary endpoints included clinical responses, breast conservation rate, safety, and discovery of molecular features associated with outcome.Results: Between 2009 and 2013, 145 patients were accrued; 36% of patients in the everolimus arm and 49% of patients in the placebo arm achieved pCR; in each arm, 50% of patients achieved complete responses by imaging. Higher rates of neutropenia, mucositis, and transaminase elevation were seen with everolimus. Clinical response to therapy and long-term outcome correlated with increased frequency of DNA damage response (DDR) gene mutations, Basal-like1 and Mesenchymal TNBC-subtypes, AR-negative status, and high Ki67, but not with tumor-infiltrating lymphocytes.Conclusions: The paclitaxel/cisplatin combination was well tolerated and active, but addition of everolimus was associated with more adverse events without improvement in pCR or clinical response. However, discoveries made from correlative studies could lead to predictive TNBC biomarkers that may impact clinical decision-making and provide new avenues for mechanistic exploration that could lead to clinical utility. Clin Cancer Res; 23(15); 4035–45. ©2017 AACR.

Supplementary Figures - PDF file 440K, Figure S1. Figure S1. Validation the IHC protocol for LDHB using FFPE MDAMB231 cells with stable knockdown of LDHA or LDHB. Reduced staining for LDHB was detected only in the LDHB knockdown line. Control cells were infected with a non-silencing shRNA. Figure S2. LDHB, when present, substantially contributed to the total LDH activity in cell lines. Figure S3. A, verification of LDHA or LDHB knockdown by LDH activity in stable isogenic cell lines, MDAMB231 and HCC1937. Figure S4. LDHB association with PAM50 intrinsic subtype in primary breast cancers. A, LDHB mRNA expression separated by intrinsic subtype: Perou GSE10893 (8) Figure S5. High LDHB was associated with triple-negative breast cancer. LDHB mRNA expression of HER2 (by IHC/FISH)-negative disease separated by clinical classification Figure S6. LDHB mRNA expression within PAM50 intrinsic subtypes for HR-positive/HER2-negative breast cancers. Figure S7. LDHB mRNA expression within PAM50 intrinsic subtypes for triple-negative breast cancers Figure S8. LDHB mRNA expression was highly associated with cell cycle proliferation marker, CCNB1, for basal-like cancers within the triple-negative group. Figure S9. Association between mRNA expression and copy number for LDHB for the breast invasive carcinoma cohort separated by (A) basal and (B) luminal A/B subtypes for TCGA "manuscript samples" within the cBio Cancer Genomics Portal

Presence of Intratumoral and Stromal Tumor-Infiltrating Lymphocytes (TILs) did not Correlate with Pathological Responses

Alterations in the PI3K/AKT/mTOR Pathway are not Enriched in Clinical Responders

Supplementary Figure 7: Changes in immunologic signatures in response to NAC are not associated with outcome in non-TNBC tumors with residual disease.

Supplementary Figure 3: The prognostic value of sTILs is primarily confined to the post-NAC specimen in TNBC patients with residual disease.

Supplementary Data - PDF file 89K, Supplementary Methods Tissue microarray (TMA) of triple-negative breast cancers

Supplementary Figure 6: Neither time of sample nor institution drive significant gene expression changes.

Supplementary Tables

Supplementary Figure 4: sTILs are not prognostic in ER+ or HER2+ disease.