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African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function. Significance: Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

In this study, we used whole-exome sequencing of a cohort of 45 advanced-stage, treatment-naïve Nigerian (NG) primary prostate cancer tumors and 11 unmatched nontumor tissues to compare genomic mutations with African American (AA) and European American (EA) The Cancer Genome Atlas (TCGA) prostate cancer. NG samples were collected from six sites in central and southwest Nigeria. After whole-exome sequencing, samples were processed using GATK best practices. BRCA1 (100%), BARD1 (45%), BRCA2 (27%), and PMS2(18%) had germline alterations in at least two NG nontumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%), and PMS2 (16%)] similar to NG samples. Of the most frequently mutated genes, BRCA1 showed a statistically (P ≤ 0.05) higher germline mutation frequency in men of African ancestry (MAA) and increasing variant frequency with increased African ancestry. Disaggregating gene-level mutation frequencies by variants revealed both ancestry-linked and NG-specific germline variant patterns. Driven by rs799917 (T>C), BRCA1 showed an increasing mutation frequency as African ancestry increased. BRCA2_rs11571831 was present only in MAA, and BRCA2_rs766173 was elevated in NG men. A total of 133 somatic variants were present in 26 prostate cancer–associated genes within the NG tumor cohort. BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%), and PMS2 (11%) showed mutation frequencies >10%. Compared with TCGA cohorts, NG tumors showed statistically significant elevated frequencies of BRCA2, APC, and BRCA1. The NG cohort variant pattern shared similarities (cosign similarities ≥0.734) with Catalogue of Somatic Mutations in Cancer signatures 5 and 6, and mutated genes showed significant (q < 0.001) gene ontology (GO) and functional enrichment in mismatch repair and non-homologous repair deficiency pathways. Here, we showed that mutations in DNA damage response genes were higher in NG prostate cancer samples and that a portion of those mutations correlate with African ancestry. Moreover, we identified variants of unknown significance that may contribute to population-specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the NG prostate cancer exome to date and highlight the need to increase diversity of study populations. Significance: MAA have higher rates of prostate cancer incidence and mortality, however, are severely underrepresented in genomic studies. This is the first study utilizing whole-exome sequencing in NG men to identify West African ancestry-linked variant patterns that impact DNA damage repair pathways.

ST_13 BAZ1A and lineage plasticity

SF_6 RNA-Seq PCA

ST_12 ChIP-Seq to RNA-Seq

SF_8 RNA-Seq GSEA

SF_13 GSEA tumors

Supplementary Table _2 - RIME II

SF_12 BAZ1A-GSEA

ST_3 ATAC-Seq

SF_3 motifs in ATAC-Seq

ST_11 ATAC- to RNA-Seq

African American (AA) prostate cancer associates with vitamin D3 deficiency, but vitamin D receptor (VDR) genomic actions have not been investigated in this context. We undertook VDR proteogenomic analyses in European American (EA) and AA prostate cell lines and four clinical cohorts. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analyses revealed that nonmalignant AA RC43N prostate cells displayed the greatest dynamic protein content in the VDR complex. Likewise, in AA cells, Assay for Transposase-Accessible Chromatin using sequencing established greater 1α,25(OH)2D3-regulated chromatin accessibility, chromatin immunoprecipitation sequencing revealed significant enhancer-enriched VDR cistrome, and RNA sequencing identified the largest 1α,25(OH)2D3-dependent transcriptome. These VDR functions were significantly corrupted in the isogenic AA RC43T prostate cancer cells, and significantly distinct from EA cell models. We identified reduced expression of the chromatin remodeler, BAZ1A, in three AA prostate cancer cohorts as well as RC43T compared with RC43N. Restored BAZ1A expression significantly increased 1α,25(OH)2D3-regulated VDR-dependent gene expression in RC43T, but not HPr1AR or LNCaP cells. The clinical impact of VDR cistrome-transcriptome relationships were tested in three different clinical prostate cancer cohorts. Strikingly, only in AA patients with prostate cancer, the genes bound by VDR and/or associated with 1α,25(OH)2D3-dependent open chromatin (i) predicted progression from high-grade prostatic intraepithelial neoplasia to prostate cancer; (ii) responded to vitamin D3 supplementation in prostate cancer tumors; (iii) differentially responded to 25(OH)D3 serum levels. Finally, partial correlation analyses established that BAZ1A and components of the VDR complex identified by RIME significantly strengthened the correlation between VDR and target genes in AA prostate cancer only. Therefore, VDR transcriptional control is most potent in AA prostate cells and distorted through a BAZ1A-dependent control of VDR function.Significance:Our study identified that genomic ancestry drives the VDR complex composition, genomic distribution, and transcriptional function, and is disrupted by BAZ1A and illustrates a novel driver for AA prostate cancer.

SF_9 overlap miRNA

ST_4 ChIP-Seq

ST_5 ChromHMM

SF_10 cistrome-transcriptome

ST_7 ATAC-Seq to RIME

ST_10 ATAC overlap ChIP

SF_5 ChIP-Seq motif

SF_2 Ancestry cell lines

ST_15 Cistrome and miRNA expression

ST_16 Cistrome and RNA-Seq in tumors

Supplementary Table_1 RIME

SF_4 VDR cistrome

ST_6 ChIP-Seq Motif

ST_8 ATAC/ChIP-Seq to splicing

SF_1 workflow

SF_7 RNA-Seq volcanos

ST_14 Serum miRNA and PCa

In this study, we used whole-exome sequencing of a cohort of 45 advanced-stage, treatment-naïve Nigerian (NG) primary prostate cancer tumors and 11 unmatched nontumor tissues to compare genomic mutations with African American (AA) and European American (EA) The Cancer Genome Atlas (TCGA) prostate cancer. NG samples were collected from six sites in central and southwest Nigeria. After whole-exome sequencing, samples were processed using GATK best practices. BRCA1 (100%), BARD1 (45%), BRCA2 (27%), and PMS2(18%) had germline alterations in at least two NG nontumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%), and PMS2 (16%)] similar to NG samples. Of the most frequently mutated genes, BRCA1 showed a statistically (P ≤ 0.05) higher germline mutation frequency in men of African ancestry (MAA) and increasing variant frequency with increased African ancestry. Disaggregating gene-level mutation frequencies by variants revealed both ancestry-linked and NG-specific germline variant patterns. Driven by rs799917 (T>C), BRCA1 showed an increasing mutation frequency as African ancestry increased. BRCA2_rs11571831 was present only in MAA, and BRCA2_rs766173 was elevated in NG men. A total of 133 somatic variants were present in 26 prostate cancer–associated genes within the NG tumor cohort. BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%), and PMS2 (11%) showed mutation frequencies >10%. Compared with TCGA cohorts, NG tumors showed statistically significant elevated frequencies of BRCA2, APC, and BRCA1. The NG cohort variant pattern shared similarities (cosign similarities ≥0.734) with Catalogue of Somatic Mutations in Cancer signatures 5 and 6, and mutated genes showed significant (q < 0.001) gene ontology (GO) and functional enrichment in mismatch repair and non-homologous repair deficiency pathways. Here, we showed that mutations in DNA damage response genes were higher in NG prostate cancer samples and that a portion of those mutations correlate with African ancestry. Moreover, we identified variants of unknown significance that may contribute to population-specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the NG prostate cancer exome to date and highlight the need to increase diversity of study populations.Significance:MAA have higher rates of prostate cancer incidence and mortality, however, are severely underrepresented in genomic studies. This is the first study utilizing whole-exome sequencing in NG men to identify West African ancestry-linked variant patterns that impact DNA damage repair pathways.

Women of sub-Saharan African descent have disproportionately higher incidence of triple-negative breast cancer (TNBC) and TNBC-specific mortality across all populations. Population studies show racial differences in TNBC biology, including higher prevalence of basal-like and quadruple-negative subtypes in African Americans (AA). However, previous investigations relied on self-reported race (SRR) of primarily U.S. populations. Due to heterogeneous genetic admixture and biological consequences of social determinants, the true association of African ancestry with TNBC biology is unclear. To address this, we conducted RNA sequencing on an international cohort of AAs, as well as West and East Africans with TNBC. Using comprehensive genetic ancestry estimation in this African-enriched cohort, we found expression of 613 genes associated with African ancestry and 2,000+ associated with regional African ancestry. A subset of African-associated genes also showed differences in normal breast tissue. Pathway enrichment and deconvolution of tumor cellular composition revealed that tumor-associated immunologic profiles are distinct in patients of African descent.Significance:Our comprehensive ancestry quantification process revealed that ancestry-associated gene expression profiles in TNBC include population-level distinctions in immunologic landscapes. These differences may explain some differences in race–group clinical outcomes. This study shows the first definitive link between African ancestry and the TNBC immunologic landscape, from an African-enriched international multiethnic cohort.See related commentary by Hamilton et al., p. 2496.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Table 1. Known Germline Variants. Supplementary Table 2. Germline Variants. Supplementary Table 3. Known Somatic Variants. Supplementary Table 4. Somatic Vars in NG Normal. Supplementary Table 5. NG WES QC Metrics. Supplementary Table 6. NG Cohort Description. Supplementary Table 7. TCGA Cohort Description. Supplementary Table 8. AA Germ Var Frequency. Supplementary Table 9. Somatic Variants. Supplementary Table 10. NG Somatic ClueGo Results.

Supplementary Table from African Ancestry–Associated Gene Expression Profiles in Triple-Negative Breast Cancer Underlie Altered Tumor Biology and Clinical Outcome in Women of African Descent

Supplementary Data from African Ancestry–Associated Gene Expression Profiles in Triple-Negative Breast Cancer Underlie Altered Tumor Biology and Clinical Outcome in Women of African Descent

Supplementary Figure 1. WES Analysis Workflow. Supplementary Figure 2. Variant Strand Bias. Supplementary Figure 3. Somatic Variant Filtering. Supplementary Figure 4. Manual Somatic Variant Inspection. Supplementary Figure 5. Genetic Admixture Analysis. Supplementary Figure 6. Comparison of somatic variant count based on normal sample usage. Supplementary Figure 7. TCGA EA Somatic Variant Lollipop Plots. Supplementary Figure 8. NG PCa Novel Somatic Variants. Supplementary Figure 9. Nigerian Germline Variant – Normal/Tumor Comparison Lollipop Plots. Supplementary Figure 10. TCGA PCa Somatic Variant COSMIC Signature Analysis.

Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer. Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer [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 P2-13-24.

Supplementary Data from Poziotinib Inhibits HER2-Mutant–Driven Therapeutic Resistance and Multiorgan Metastasis in Breast Cancer

XLSX file - 125K, Drugs with 40-100% inhibition.

Supplementary Table S7. Univariate and multivariate Cox proportional hazard regression models of the TCGA (A), Lindgren (B), Sjodahl (C), and Kim (D), data sets using CPT1B gene expression. Univariate Cox proportional hazard regression analysis showed that CPT1B expression yields a Hazard ratios below 1 in TCGA, Lindgren, and Sjodahl, and is significantly associated with survival in TCGA cohort. The multivariate models after adjustment for other factors such as sex, age and tumor stage, revealed that low (HR less than 1) CPT1B expression was significantly associated with an increased risk of death in TCGA. Hazard ratios (HRs) derived from the Cox proportional hazards models are expressed with 95% confidence intervals (CIs) and p-values. For all analyses, p-value

XLSX file - 165K, Response of Panc502 to 125 anti-neoplastic drugs.

Supplementary Table S8. Patient characteristics of bladder specimens used for figure 4B, 4C, and 4G.

Supplement Table S5. List of differential lipids and their classification.

PDF file - 116K, In vivo response of xenograftted tumors.

PDF file - 129K

Purpose: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells.Experimental Design: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts.Results: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)–approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs.Conclusion: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. Clin Cancer Res; 19(5); 1139–46. ©2012 AACR.

The pan-HER tyrosine kinase inhibitor (TKI) neratinib is therapeutically active against metastatic breast cancers harboring activating HER2 mutations, but responses are variable and often not durable. Here we demonstrate that recurrent HER2 mutations have differential effects on endocrine therapy responsiveness, metastasis, and pan-HER TKI therapeutic sensitivity. The prevalence and prognostic significance may also depend on whether the HER2 mutant has arisen in the context of lobular versus ductal histology. The most highly recurrent HER2 mutant, L755S, was particularly resistant to neratinib but sensitive to the pan-HER TKI poziotinib, alone or in combination with fulvestrant. Poziotinib reduced tumor growth, diminished multiorgan metastasis, and inhibited mTOR activation more effectively than neratinib. Similar therapeutic effects of poziotinib were observed in both an engineered HER2L755S MCF7 model and a patient-derived xenograft harboring a HER2G778_P780dup mutation. Overall, these findings support the need for clinical evaluation of poziotinib for the treatment of HER2-mutant metastatic breast cancer.Significance:Evaluation of the functional impact of HER2 mutations on therapy-induced resistance and metastasis identifies robust antitumor activity of poziotinib and supports the clinical evaluation of poziotinib in ER+ HER2 mutant breast cancer.

Supplementary Table S3. Patient characteristics of bladder specimens used for the metabolomics and lipidomics profiling.

Supplementary Figure S6. Survival analysis of individual downregulated genes showed poor prognosis associated with CPT1B and PIGB in the Lindgren and Sjodahl cohorts; PLA2G4A in the Lindgren cohort; PIGV in the Kim and Sjodahl cohorts; ATP8B1 in the Kim and Sjodahl cohorts; INPP5D in the Kim and Sjodahl cohorts; PIGZ in the Kim and Sjodahl cohorts; PLCD3 in the Sjodahl cohort; and PLA2G10 in the Kim cohort.

PDF file - 87K, Breeding strategy for production and maintenance of the SCID hprt-null mouse.