Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

BACKGROUND: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. RESULTS: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. CONCLUSIONS: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

The breast is yet to come: current and future utility of circulating tumour DNA in breast cancer.

Advances in genomic strategies and the development of targeted therapies have enabled precision medicine to revolutionise the field of oncology. Precision medicine uses patient-specific genetic and molecular information, traditionally obtained from tumour biopsy samples, to classify tumours and treat them accordingly. However, biopsy samples often fail to provide complete tumour profiling, and the technique is expensive and, of course, relatively invasive. Advances in genomic techniques have led to improvements in the isolation and detection of circulating tumour DNA (ctDNA), a component of a peripheral blood draw/liquid biopsy. Liquid biopsy offers a minimally invasive method to gather genetic information that is representative of a global snapshot of both primary and metastatic sites and can thereby provide invaluable information for potential targeted therapies and methods for tumour surveillance. However, a lack of prospective clinical trials showing direct patient benefit has limited the implementation of liquid biopsies in standard clinical applications. Here, we review the potential of ctDNA obtained by liquid biopsy to revolutionise personalised medicine and discuss current applications of ctDNA both at the benchtop and bedside.

Circulating tumor DNA in early-stage breast cancer: new directions and potential clinical applications.

The use of circulating tumor DNA (ctDNA) in liquid biopsy as a biomarker is becoming the new paradigm for the screening and surveillance of breast and many other cancers. Liquid biopsies provide prognostic and predictive information without the limitations of tissue biopsies. Most early studies of the use of ctDNA focused on metastatic disease. However, recent advancements in ctDNA technologies have improved sensitivity and selectivity, allowing ctDNA to be detected in early-stage disease, including early-stage breast cancer. Despite a clear potential for utility, the implementation of ctDNA liquid biopsy in standard of care is significantly lacking. Researchers and clinicians are currently working to validate the clinical utility of ctDNA in diagnostics, prognostics, the surveillance of minimal residual disease, and the monitoring of therapeutic response. This review summarizes the current applications of ctDNA in early-stage breast cancer and discusses its potential uses in clinical practice.

TrkA overexpression in non-tumorigenic human breast cell lines confers oncogenic and metastatic properties.

BACKGROUND/PURPOSE: TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS: To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS: TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS: Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.

Undetectable Tumor Cell-Free DNA in a Patient With Metastatic Breast Cancer With Complete Response and Long-Term Remission.

The ability to serially monitor tumor-derived cell-free DNA (cfDNA) brings with it the potential to measure response to anticancer therapies and detect minimal residual disease (MRD). This report describes a patient with HER2-positive metastatic breast cancer with an exceptional response to trastuzumab and nab-paclitaxel who remains in complete remission several years after cessation of therapy. Next-generation sequencing of the patient's primary tumor tissue showed several mutations, including an oncogenic hotspot PIK3CA mutation. A sample of cfDNA was collected 6 years after her last therapy and then analyzed for mutant PIK3CA using digital PCR. No detectable mutations associated with the primary tumor were found despite assaying >10,000 genome equivalents, suggesting that the patient had achieved a molecular remission. Results of this case study suggest that serial monitoring of MRD using liquid biopsies could provide a useful method for individualizing treatment plans for patients with metastatic disease with extreme responses to therapy. However, large-scale clinical studies are needed to validate and implement these techniques for patient care.

The estrogen receptor-alpha S118P variant does not affect breast cancer incidence or response to endocrine therapies.

PURPOSE: Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. METHODS: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. RESULTS: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. CONCLUSIONS: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability.

The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

HER2 missense mutations have distinct effects on oncogenic signaling and migration.

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

PIK3CA mutations and TP53 alterations cooperate to increase cancerous phenotypes and tumor heterogeneity.

BACKGROUND/PURPOSE: The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. EXPERIMENTAL DESIGN/METHODS: We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. RESULTS: Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. CONCLUSIONS: This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers.

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

A phosphoproteomic screen demonstrates differential dependence on HER3 for MAP kinase pathway activation by distinct PIK3CA mutations.

The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. Conclusions: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

ABTRACT: Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

CHIP Happens: Clonal Hematopoiesis of Indeterminate Potential and Its Relationship to Solid Tumors.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the expansion of hematopoietic cells harboring leukemia-associated somatic mutations in otherwise healthy people and occurs in at least 10% of adults over 70. It is well established that people with CHIP have increased rates of hematologic malignancy, increased risk of cardiovascular disease, and worse all-cause mortality compared with those without CHIP. Despite recent advancements in understanding CHIP as it relates to these known outcomes, much remains to be learned about the development and role of CHIP in other disease states. Emerging research has identified high rates of CHIP in patients with solid tumors, driven in part by oncologic therapy, and revealed associations between CHIP and differential outcomes in both solid tumors and other diseases. Recent studies have demonstrated that CHIP can contribute to dysregulated inflammatory signaling in multiple contexts, underscoring the importance of interrogating how CHIP might alter tumor immunology. Here, we review the role of CHIP mutations in clonal expansion of hematopoietic cells, explore the relationship between CHIP and solid tumors, and discuss the potential roles of CHIP in inflammation and solid tumor biology.

NOTCH1 PEST domain variants are responsive to standard of care treatments despite distinct transformative properties in a breast cancer model.

Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications.

Single cell analysis of cribriform prostate cancer reveals cell intrinsic and tumor microenvironmental pathways of aggressive disease.

Cribriform prostate cancer, found in both invasive cribriform carcinoma (ICC) and intraductal carcinoma (IDC), is an aggressive histological subtype that is associated with progression to lethal disease. To delineate the molecular and cellular underpinnings of ICC/IDC aggressiveness, this study examines paired ICC/IDC and benign prostate surgical samples by single-cell RNA-sequencing, TCR sequencing, and histology. ICC/IDC cancer cells express genes associated with metastasis and targets with potential for therapeutic intervention. Pathway analyses and ligand/receptor status model cellular interactions among ICC/IDC and the tumor microenvironment (TME) including JAG1/NOTCH. The ICC/IDC TME is hallmarked by increased angiogenesis and immunosuppressive fibroblasts (CTHRC1+ASPN+FAP+ENG+) along with fewer T cells, elevated T cell dysfunction, and increased C1QB+TREM2+APOE+-M2 macrophages. These findings support that cancer cell intrinsic pathways and a complex immunosuppressive TME contribute to the aggressive phenotype of ICC/IDC. These data highlight potential therapeutic opportunities to restore immune signaling in patients with ICC/IDC that may afford better outcomes.

Demographics, Outcomes, and Risk Factors for Patients with Sarcoma and COVID-19: A CCC19-Registry Based Retrospective Cohort Study.

BACKGROUND: Patients with sarcoma often require individualized treatment strategies and are likely to receive aggressive immunosuppressive therapies, which may place them at higher risk for severe COVID-19. We aimed to describe demographics, risk factors, and outcomes for patients with sarcoma and COVID-19. METHODS: We performed a retrospective cohort study of patients with sarcoma and COVID-19 reported to the COVID-19 and Cancer Consortium (CCC19) registry (NCT04354701) from 17 March 2020 to 30 September 2021. Demographics, sarcoma histologic type, treatments, and COVID-19 outcomes were analyzed. RESULTS: of 281 patients, 49% (n = 139) were hospitalized, 33% (n = 93) received supplemental oxygen, 11% (n = 31) were admitted to the ICU, and 6% (n = 16) received mechanical ventilation. A total of 23 (8%) died within 30 days of COVID-19 diagnosis and 44 (16%) died overall at the time of analysis. When evaluated by sarcoma subtype, patients with bone sarcoma and COVID-19 had a higher mortality rate than patients from a matched SEER cohort (13.5% vs 4.4%). Older age, poor performance status, recent systemic anti-cancer therapy, and lung metastases all contributed to higher COVID-19 severity. CONCLUSIONS: Patients with sarcoma have high rates of severe COVID-19 and those with bone sarcoma may have the greatest risk of death.

Systemic inhibition of PTPN22 augments anticancer immunity.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

Hierarchical tumor heterogeneity mediated by cell contact between distinct genetic subclones.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.