Are FGFR Fusions and Mutations the Next Tumor-Agnostic Targets in Oncology?

Gong et al present two NCI-MATCH tumor-agnostic trials evaluating erdafitinib for FGFR-altered cancers, marking steppingstones in precision oncology.

FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

The Irreversible FGFR Inhibitor KIN-3248 Overcomes FGFR2 Kinase Domain Mutations.

PURPOSE: FGFR2 and FGFR3 show oncogenic activation in many cancer types, often through chromosomal fusion or extracellular domain mutation. FGFR2 and FGFR3 alterations are most prevalent in intrahepatic cholangiocarcinoma (ICC) and bladder cancers, respectively, and multiple selective reversible and covalent pan-FGFR tyrosine kinase inhibitors (TKI) have been approved in these contexts. However, resistance, often due to acquired secondary mutations in the FGFR2/3 kinase domain, limits efficacy. Resistance is typically polyclonal, involving a spectrum of different mutations that most frequently affect the molecular brake and gatekeeper residues (N550 and V565 in FGFR2). EXPERIMENTAL DESIGN: Here, we characterize the activity of the next-generation covalent FGFR inhibitor, KIN-3248, in preclinical models of FGFR2 fusion+ ICC harboring a series of secondary kinase domain mutations, in vitro and in vivo. We also test select FGFR3 alleles in bladder cancer models. RESULTS: KIN-3248 exhibits potent selectivity for FGFR1-3 and retains activity against various FGFR2 kinase domain mutations, in addition to being effective against FGFR3 V555M and N540K mutations. Notably, KIN-3248 activity extends to the FGFR2 V565F gatekeeper mutation, which causes profound resistance to currently approved FGFR inhibitors. Combination treatment with EGFR or MEK inhibitors potentiates KIN-3248 efficacy in vivo, including in models harboring FGFR2 kinase domain mutations. CONCLUSIONS: Thus, KIN-3248 is a novel FGFR1-4 inhibitor whose distinct activity profile against FGFR kinase domain mutations highlights its potential for the treatment of ICC and other FGFR-driven cancers.

Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations.

PIK3CA mutations occur in ∼8% of cancers, including ∼40% of HR-positive breast cancers, where the PI3K-alpha (PI3Kα)-selective inhibitor alpelisib is FDA approved in combination with fulvestrant. Although prior studies have identified resistance mechanisms, such as PTEN loss, clinically acquired resistance to PI3Kα inhibitors remains poorly understood. Through serial liquid biopsies and rapid autopsies in 39 patients with advanced breast cancer developing acquired resistance to PI3Kα inhibitors, we observe that 50% of patients acquire genomic alterations within the PI3K pathway, including PTEN loss and activating AKT1 mutations. Notably, although secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Kα inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket. Some mutations had differential effects on PI3Kα-selective versus pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Kα-inhibitor RLY-2608. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. SIGNIFICANCE: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

Landscape of Clinical Resistance Mechanisms to FGFR Inhibitors in FGFR2-Altered Cholangiocarcinoma.

PURPOSE: FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. EXPERIMENTAL DESIGN: We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. RESULTS: Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. CONCLUSIONS: These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

KRAS Wild-Type Pancreatic Cancer: Decoding Genomics, Unlocking Therapeutic Potential.

In a landscape dominated by pivotal KRAS mutations, there has been limited exploration of KRAS wild-type pancreatic cancer. A recent study highlights other mitogen-activated kinase pathway alterations as alternative drivers in these tumors, which holds the key to unlocking a realm of targeted therapies for patients with this understudied cancer subtype. See related article by Singh et al., p. 4627.

Comparative Effectiveness of Immune Checkpoint Inhibitors vs Chemotherapy in Patients With Metastatic Colorectal Cancer With Measures of Microsatellite Instability, Mismatch Repair, or Tumor Mutational Burden.

Importance: The KEYNOTE-177 trial demonstrated that patients with metastatic colorectal cancer (MCRC) with high microsatellite instability (MSI-H) and/or mismatch repair deficiency (DMMR) have better outcomes when receiving first-line immune checkpoint inhibitors (ICIs) compared with chemotherapy. Data on performance of ICIs in patients with MCRC in standard practice settings remain limited, and direct MMR vs MSI outcome association comparisons are lacking. Objective: To validate MSI (determined by next-generation sequencing [NGS]) as a biomarker of ICI effectiveness among patients with MCRC in standard practice settings and examine the association of MSI assessed by NGS, DMMR by immunohistochemistry, and tumor mutational burden (cutoff, 10 mutations/megabase) with ICI outcomes. Design, Setting, and Participants: This comparative effectiveness research study of outcomes in prospectively defined biomarker subgroups used data from a deidentified clinicogenomic database and included patients who received Foundation Medicine testing (FoundationOne or FoundationOne CDx) during routine clinical care at approximately 280 US academic or community-based cancer clinics between March 2014 and December 2021. The population included 1 cohort of patients with MSI-H MCRC who received first-line ICIs or chemotherapy and a second cohort who received ICIs in any line of therapy (LOT) for biomarker examination. Exposures: ICI therapy or chemotherapy assigned at physician discretion without randomization. Main Outcomes and Measures: The main outcomes were time to next treatment (TTNT), progression-free survival (PFS), and overall survival (OS). Hazard ratios were adjusted for known prognostic imbalances. Comparisons of explanatory power used the likelihood ratio test. Results: A total of 138 patients (median age, 67.0 years [IQR, 56.2-74.0 years]; 73 [52.9%] female) with MSI-H MCRC received first-line ICIs or chemotherapy. A total of 182 patients (median age, 64.5 years [IQR, 55.2-72.0]; 98 [53.8%] female) received ICIs in any LOT. Patients receiving first-line ICIs vs chemotherapy had longer TTNT (median, not reached [NR] vs 7.23 months [IQR, 6.21-9.72 months]; adjusted hazard ratio [AHR], 0.17; 95% CI, 0.08-0.35; P < .001), PFS (median, 24.87 months [IQR, 19.10 months to NR] vs 5.65 months [IQR, 4.70-8.34 months]; AHR, 0.31; 95% CI, 0.18-0.52; P < .001), and OS (median, NR vs 24.1 months [IQR, 13.90 months to NR]; HR, 0.45; 95% CI, 0.23-0.88; P = .02). MSI added to DMMR better anticipated TTNT and PFS in patients receiving ICIs than DMMR alone. The same was not observed when DMMR evaluation was added to MSI. Conclusions and Relevance: In this comparative effectiveness research study, MSI assessed by NGS robustly identified patients with favorable outcomes on first-line ICIs vs chemotherapy and appeared to better anticipate ICI outcomes compared with DMMR.

Emerging Role of Targeted Therapy in Metastatic Pancreatic Adenocarcinoma.

The aggressive biology of pancreatic ductal adenocarcinoma (PDAC), along with its limited sensitivity to many systemic therapies, presents a major challenge in the management of patients with metastatic PDAC. Over the past decade, the incorporation of combinatorial cytotoxic chemotherapy regimens has improved patient outcomes. Despite these advances, resistance to cytotoxic chemotherapy inevitably occurs, and there is a great need for effective therapies. A major focus of research has been to identify molecularly defined subpopulations of patients with PDAC who may benefit from targeted therapies that are matched to their molecular profile. Recent successes include the demonstration of the efficacy of maintenance PARP inhibition in PDAC tumors harboring deleterious BRCA1, BRCA2, and PALB2 alterations. In addition, while therapeutic targeting of KRAS was long thought to be infeasible, emerging data on the efficacy of KRAS G12C inhibitors have increased optimism about next-generation KRAS-directed therapies in PDAC. Meanwhile, KRAS wild-type PDAC encompasses a unique molecular subpopulation of PDAC that is enriched for targetable genetic alterations, such as oncogenic BRAF alterations, mismatch repair deficiency, and FGFR2, ALK, NTRK, ROS1, NRG1, and RET rearrangements. As more molecularly targeted therapies are developed, precision medicine has the potential to revolutionize the treatment of patients with metastatic PDAC.

Effective Treatment of Established Bone Metastases Can Be Achieved by Combinatorial Osteoclast Blockade and Depletion of Granulocytic Subsets.

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)-heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers-originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow-resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro Anti-Gr1-mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

Cell-free DNA alterations in the AR enhancer and locus predict resistance to AR-directed therapy in patients with metastatic prostate cancer.

PURPOSE: Cell-free DNA (cfDNA) and circulating tumor cell (CTC) based liquid biopsies have emerged as potential tools to predict responses to androgen receptor (AR)-directed therapy in metastatic prostate cancer. However, due to complex mechanisms and incomplete understanding of genomic events involved in metastatic prostate cancer resistance, current assays (e.g. CTC AR-V7) demonstrate low sensitivity and remain underutilized. The recent discovery of AR enhancer amplification in >80% of metastatic patients and its association with disease resistance presents an opportunity to improve upon current assays. We hypothesized that tracking AR/enhancer genomic alterations in plasma cfDNA would detect resistance with high sensitivity and specificity. METHODS: We developed a targeted sequencing and analysis method as part of a new assay called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq). We applied EnhanceAR-Seq to plasma collected from 40 patients with metastatic prostate cancer treated with AR-directed therapy to monitor AR/enhancer genomic alterations and correlate these events with therapy resistance, progression-free survival (PFS) and overall survival (OS). RESULTS: EnhanceAR-Seq identified genomic alterations in the AR/enhancer locus in 45% of cases, including a 40% rate of AR enhancer amplification. Patients with AR/enhancer alterations had significantly worse PFS and OS than those without (6-month PFS: 30% vs. 71%, P=0.0002; 6-month OS: 59% vs. 100%, P=0.0015). AR/enhancer alterations in plasma cfDNA detected 18 of 23 resistant cases (78%) and outperformed the CTC AR-V7 assay which was also run on a subset of patients. CONCLUSION: cfDNA-based AR locus alterations, including of the enhancer, are strongly associated with resistance to AR-directed therapy and significantly worse survival. cfDNA analysis using EnhanceAR-Seq may enable more precise risk stratification and personalized therapeutic approaches for metastatic prostate cancer.

PI3K Inhibitors in Breast Cancer Therapy.

PURPOSE OF REVIEW: The phosphatidylinositol 3-kinase (PI3K) pathway is the most common aberrantly activated pathway in breast cancer, making it an attractive therapeutic target. In this review, we will discuss the rationale for targeting PI3K/AKT signaling and the development of PI3K/AKT inhibitors in breast cancer. RECENT FINDINGS: Although the initial clinical trials with pan-PI3K inhibitors were challenged by high toxicities and modest antitumor effect, there has been continued effort to develop agents more precisely targeting PI3K isoforms to improve therapeutic index. Alpelisib in combination with fulvestrant is now available in the clinic for postmenopausal women with advanced or metastatic hormone receptor (HR)-positive, HER2-negative, PIK3CA-mutated breast cancer. In addition, promising data has been observed in randomized phase II trials of AKT inhibitors in combination with fulvestrant or paclitaxel in metastatic HR-positive, HER2-negative disease and triple negative breast cancer (TNBC), respectively. The high frequency of genetic alterations in the PI3K pathway has provided the rationale for development of inhibitors targeting PI3K/AKT. Despite initial disappointment with several randomized trials of pan-PI3K inhibitors in HR-positive breast cancer, there has been continued effort to more precisely target PI3K isoforms, which has led to clinical benefit for patients with advanced breast cancer.

Extended adjuvant endocrine therapy in hormone-receptor-positive early breast cancer.

PURPOSE OF REVIEW: The risk of relapse associated with oestrogen receptor-positive early breast cancer persists for at least 15 years after diagnosis. Several large clinical trials have examined extended adjuvant endocrine therapy. RECENT FINDINGS: The MA.17 trial demonstrated improved disease-free survival (DFS) with use of letrozole for 5 years after some years of tamoxifen and an overall survival advantage for this approach in women with node-positive oestrogen receptor-positive cancer at diagnosis. The subsequent adjuvant tamoxifen - to offer more? and adjuvant tamoxifen: longer against shorter trials demonstrated a DFS advantage for 10 years of tamoxifen over 5 years. The recently reported MA.17R trial randomized women who had already completed 5 years of aromatase inhibitor therapy with or without previous tamoxifen to further 5 years of letrozole or placebo. DFS was significantly improved in the extended letrozole group, quality of life was similar but bone fracture rates were higher. The absolute benefit in terms of reduced distant recurrences in these studies is modest, and tolerability and compliance challenges remain. SUMMARY: Physicians and patients now have multiple evidence-based treatment options for women who complete 5 years of adjuvant endocrine therapy. Extended therapy with either tamoxifen or letrozole should be considered for all and decision based on menopausal status, individual risk, tolerance and magnitude of potential benefit.

PDK1-SGK1 Signaling Sustains AKT-Independent mTORC1 Activation and Confers Resistance to PI3Kα Inhibition.

PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells.

PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor-positive breast cancer.

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor.

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110ß blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

MACC1 expression levels as a novel prognostic marker for colorectal cancer.

Metastasis-associated in colon cancer-1 (MACC1) is key in promoting tumor proliferation and invasion, and is mediated by the hepatocyte growth factor (HGF) and mesenchymal-epithelial transition factor. Previous reports have revealed that MACC1 is a novel oncogene that is expressed in various types of gastrointestinal cancer. The present study comprised of 174 patients who underwent curative surgery for colorectal cancer (CRC). The correlation between gene expression and clinical parameters of the patients was assessed. It was identified that patients exhibiting high MACC1 expression levels were statistically more susceptible to distant metastases and a poor prognosis, and those exhibiting low MACC1 expression showed improved disease-free and overall survival than those with high expression. Therefore, the present data indicates that MACC1 expression levels may present as a prognostic factor in CRC patients.

Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.