RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

PURPOSE: ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. EXPERIMENTAL DESIGN: Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. RESULTS: ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. CONCLUSIONS: Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Keeping It in the Family: HER3 as a Target in Brain Metastases.

In 180 patients with metastatic breast cancer and non-small cell lung cancer (NSCLC), HER3 expression was found in >70% of brain metastases (BM). HER3-targeting antibody-drug conjugates have demonstrated efficacy in HER3-expressing metastatic breast cancer and NSCLC. Thus, HER3 expression by IHC may be a biomarker for development of HER3-targeting BM-specific therapeutics. See related article by Tomasich et al., p. 3225.

PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Preclinical and Clinical Efficacy of Trastuzumab Deruxtecan in Breast Cancer Brain Metastases.

PURPOSE: Brain metastases can occur in up to 50% of patients with metastatic HER2-positive breast cancer. Because patients with active brain metastases were excluded from previous pivotal clinical trials, the central nervous system (CNS) activity of the antibody-drug conjugate trastuzumab deruxtecan (T-DXd) is not well characterized. EXPERIMENTAL DESIGN: We studied how T-DXd affects growth and overall survival in orthotopic patient-derived xenografts (PDX) of HER2-positive and HER2-low breast cancer brain metastases (BCBM). Separately, we evaluated the effects of T-DXd in a retrospective cohort study of 17 patients with stable or active brain metastases. RESULTS: T-DXd inhibited tumor growth and prolonged survival in orthotopic PDX models of HER2-positive (IHC 3+) and HER2-low (IHC 2+/FISH ratio < 2) BCBMs. T-DXd reduced tumor size and prolonged survival in a T-DM1-resistant HER2-positive BCBM PDX model. In a retrospective multi-institutional cohort study of 17 patients with predominantly HER2-positive BCBMs, the CNS objective response rate (ORR) was 73% (11/15) while extracranial response rate was 45% (5/11). In the subset of patients with untreated or progressive BCBM at baseline, the CNS ORR was 70% (7/10). The median time on treatment with T-DXd was 8.9 (1.3-16.2) months, with 42% (7/17) remaining on treatment at data cutoff. CONCLUSIONS: T-DXd demonstrates evidence of CNS activity in HER2-positive and HER2-low PDX models of BCBM and preliminary evidence of clinical efficacy in a multi-institution case series of patients with BCBM. Prospective clinical trials to further evaluate CNS activity of T-DXd in patients with active brain metastases are warranted. See related commentary by Soffietti and Pellerino, p. 8.

STING agonism reprograms tumor-associated macrophages and overcomes resistance to PARP inhibition in BRCA1-deficient models of breast cancer.

PARP inhibitors (PARPi) have drastically changed the treatment landscape of advanced ovarian tumors with BRCA mutations. However, the impact of this class of inhibitors in patients with advanced BRCA-mutant breast cancer is relatively modest. Using a syngeneic genetically-engineered mouse model of breast tumor driven by Brca1 deficiency, we show that tumor-associated macrophages (TAMs) blunt PARPi efficacy both in vivo and in vitro. Mechanistically, BRCA1-deficient breast tumor cells induce pro-tumor polarization of TAMs, which in turn suppress PARPi-elicited DNA damage in tumor cells, leading to reduced production of dsDNA fragments and synthetic lethality, hence impairing STING-dependent anti-tumor immunity. STING agonists reprogram M2-like pro-tumor macrophages into an M1-like anti-tumor state in a macrophage STING-dependent manner. Systemic administration of a STING agonist breaches multiple layers of tumor cell-mediated suppression of immune cells, and synergizes with PARPi to suppress tumor growth. The therapeutic benefits of this combination require host STING and are mediated by a type I IFN response and CD8+ T cells, but do not rely on tumor cell-intrinsic STING. Our data illustrate the importance of targeting innate immune suppression to facilitate PARPi-mediated engagement of anti-tumor immunity in breast cancer.

p16INK4A-deficiency predicts response to combined HER2 and CDK4/6 inhibition in HER2+ breast cancer brain metastases.

Approximately 50% of patients with metastatic HER2-positive (HER2+) breast cancer develop brain metastases (BCBMs). We report that the tumor suppressor p16INK4A is deficient in the majority of HER2+ BCBMs. p16INK4A-deficiency as measured by protein immunohistochemistry predicted response to combined tucatinib and abemaciclib in orthotopic patient-derived xenografts (PDXs) of HER2 + BCBMs. Our findings establish the rationale for a biomarker-driven clinical trial of combined CDK4/6- and HER2-targeted agents for patients with HER2 + BCBM.

Temporal and spatial topography of cell proliferation in cancer.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

Integrating Immunotherapy and Targeted Therapy in Cancer Treatment: Mechanistic Insights and Clinical Implications.

Small-molecule targeted therapies have demonstrated outstanding potential in the clinic. These drugs are designed to minimize adverse effects by selectively attacking cancer cells while exerting minimal damage to normal cells. Although initial response to targeted therapies may be high, yielding positive response rates and often improving survival for an important percentage of patients, resistance often limits long-term effectiveness. On the other hand, immunotherapy has demonstrated durable results, yet for a limited number of patients. Growing evidence indicates that some targeted agents can modulate different components of the antitumor immune response. These include immune sensitization by inhibiting tumor cell-intrinsic immune evasion programs or enhancing antigenicity, as well as direct effects on immune effector and immunosuppressive cells. The combination of these two approaches, therefore, has the potential to result in synergistic and durable outcomes for patients. In this review, we focus on the latest advances on integrating immunotherapy with small-molecule targeted inhibitors. In particular, we discuss how specific oncogenic events differentially affect immune response, and the implications of these findings on the rational design of effective combinations of immunotherapy and targeted therapies.

AKT1low quiescent cancer cells in ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast precedes the development of invasive breast cancer and reflects the genomic changes and protein expression profile of invasive disease. AKT1low cancer cells (QCC) are a rare, drug-tolerant, epigenetically plastic, and quiescent cancer cell subset that we previously identified at a frequency of 0.5-1% in primary breast tumors using the marker profile: AKTlow/H3K9me2low/HES1high. Here we used quantitative immunofluorescence microscopy with computational image analysis to show that AKT1low QCCs are present in DCIS from patients with and without co-existing invasive breast cancer. These data suggest that a drug-resistant, quiescent cancer cell state is present in premalignant breast lesions prior to the development of invasive disease. These findings warrant further study of whether AKT1low QCCs contribute to invasive tumor development and recurrence, similar to their role in more advanced malignancy.

Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.

Breast cancer brain metastasis (BCBM) remains a major clinical problem. Approximately 10-16% of patients with breast cancer develop brain metastases (BCBM). However, no systemic therapy has gained regulatory approval for the specific treatment of BCBM and this remains an area of persistent, unmet medical need. Rapid, predictive and clinically-relevant animal models are critical to study the biology of brain metastases and to identify effective therapeutic approaches for patients with BCBM. Here, we describe a method for efficient establishment of orthotopic mouse models of patient-derived brain metastases via an improved intracarotid injection protocol that permits tumor cell growth in the unique brain microenvironment without compromising the blood-brain barrier (BBB). We demonstrate that our newly improved models of patient-derived brain metastases recapitulate the histologic, molecular, and genetic characteristics of their matched patient tumor specimens and thus represent a potentially powerful tool for pre-clinical and translational research.

Drug Resistance in HER2-Positive Breast Cancer Brain Metastases: Blame the Barrier or the Brain?

The brain is the most common site of first metastasis for patients with HER2-positive breast cancer treated with HER2-targeting drugs. However, the development of effective therapies for breast cancer brain metastases (BCBM) is limited by an incomplete understanding of the mechanisms governing drug sensitivity in the central nervous system. Pharmacodynamic data from patients and in vivo models suggest that inadequate drug penetration across the "blood-tumor" barrier is not the whole story. Using HER2-positive BCBMs as a case study, we highlight recent data from orthotopic brain metastasis models that implicate brain-specific drug resistance mechanisms in BCBMs and suggest a translational research paradigm to guide drug development for treatment of BCBMs. Clin Cancer Res; 24(8); 1795-804. ©2018 AACR.

AKT1low Quiescent Cancer Cells Promote Solid Tumor Growth.

Human tumor growth depends on rapidly dividing cancer cells driving population expansion. Even advanced tumors, however, contain slowly proliferating cancer cells for reasons that remain unclear. Here, we selectively disrupt the ability of rapidly proliferating cancer cells to spawn AKT1low daughter cells that are rare, slowly proliferating, tumor-initiating, and chemotherapy-resistant, using ß1-integrin activation and the AKT1-E17K-mutant oncoprotein as experimental tools in vivo Surprisingly, we find that selective depletion of AKT1low slow proliferators actually reduces the growth of a molecularly diverse panel of human cancer cell xenograft models without globally altering cell proliferation or survival in vivo Moreover, we find that unusual cancer patients with AKT1-E17K-mutant solid tumors also fail to produce AKT1low quiescent cancer cells and that this correlates with significantly prolonged survival after adjuvant treatment compared with other patients. These findings support a model whereby human solid tumor growth depends on not only rapidly proliferating cancer cells but also on the continuous production of AKT1low slow proliferators. Mol Cancer Ther; 17(1); 254-63. ©2017 AACR.

AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer.

BACKGROUND: Absence of pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) correlates with poor long-term survival in patients with triple negative breast cancer (TNBC). These incomplete treatment responses are likely determined by mechanisms that enable cancer cells to resist being killed. However, the detailed characterization of a drug-resistant cancer cell state in residual TNBC tissue after NACT has remained elusive. AKT1low quiescent cancer cells (QCCs) are a quiescent, epigenetically plastic, and chemotherapy-resistant subpopulation initially identified in experimental cancer models. Here, we asked whether QCCs exist in primary tumors from patients with TNBC and persist after treatment with NACT. METHODS: We obtained pre-treatment biopsy, post-treatment mastectomy, and metastatic specimens from a retrospective cohort of TNBC patients treated with NACT at Massachusetts General Hospital (n = 25). Using quantitative automated immunofluorescence microscopy, QCCs were identified as AKTlow/H3K9me2low/HES1high cancer cells using prespecified immunofluorescence intensity thresholds. QCCs were represented in 2D and 3D digital tumor maps and QCC percentage (QCC-P) and QCC cluster index (QCC-CI) were determined for each sample. RESULTS: We showed that QCCs exist as non-random and heterogeneously distributed clusters within primary breast tumors. In addition, these QCC clusters persist after treatment with multi-agent, multi-cycle, neoadjuvant chemotherapy in both residual primary tumors and nodal and distant metastases in patients with triple negative breast cancer. CONCLUSIONS: These first-in-human data potentially qualify AKT1low quiescent cancer cells as a non-genetic cell state that persists after neoadjuvant chemotherapy in triple negative breast cancer patients and warrants further study.

JARID1B Enables Transit between Distinct States of the Stem-like Cell Population in Oral Cancers.

The degree of heterogeneity among cancer stem cells (CSC) remains ill-defined and may hinder effective anti-CSC therapy. Evaluation of oral cancers for such heterogeneity identified two compartments within the CSC pool. One compartment was detected using a reporter for expression of the H3K4me3 demethylase JARID1B to isolate a JARID1B(high) fraction of cells with stem cell-like function. JARID1B(high) cells expressed oral CSC markers including CD44 and ALDH1 and showed increased PI3K pathway activation. They were distinguished from a fraction in a G0-like cell-cycle state characterized by low reactive oxygen species and suppressed PI3K/AKT signaling. G0-like cells lacked conventional CSC markers but were primed to acquire stem cell-like function by upregulating JARID1B, which directly mediated transition to a state expressing known oral CSC markers. The transition was regulated by PI3K signals acting upstream of JARID1B expression, resulting in PI3K inhibition depleting JARID1B(high) cells but expanding the G0-like subset. These findings define a novel developmental relationship between two cell phenotypes that may jointly contribute to CSC maintenance. Expansion of the G0-like subset during targeted depletion of JARID1B(high) cells implicates it as a candidate therapeutic target within the oral CSC pool. Cancer Res; 76(18); 5538-49. ©2016 AACR.

ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer.

PURPOSE: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of non-small cell lung cancer (NSCLC). Recently, several case reports and small series have reported that ALK rearrangements can overlap with other oncogenic drivers in NSCLC in crizotinib-naïve and crizotinib-resistant cancers. EXPERIMENTAL DESIGN: We reviewed clinical genotyping data from 1,683 patients with NSCLC and investigated the prevalence of concomitant EGFR or KRAS mutations among patients with ALK-positive NSCLC. We also examined biopsy specimens from 34 patients with ALK-positive NSCLC after the development of resistance to crizotinib. RESULTS: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. Four patients with KRAS mutations were found to have abnormal ALK FISH patterns, most commonly involving isolated 5' green probes. Sufficient tissue was available for confirmatory ALK immunohistochemistry in 3 cases, all of which were negative for ALK expression. Among patients with ALK-positive NSCLC who acquired resistance to crizotinib, repeat biopsy specimens were ALK FISH positive in 29 of 29 (100%) cases. Secondary mutations in the ALK kinase domain and ALK gene amplification were observed in 7 of 34 (20.6%) and 3 of 29 (10.3%) cases, respectively. No EGFR or KRAS mutations were identified among any of the 25 crizotinib-resistant, ALK-positive patients with sufficient tissue for testing. CONCLUSIONS: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK-positive cancers with acquired resistance to crizotinib.

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

CPEB1 regulates beta-catenin mRNA translation and cell migration in astrocytes.

A crucial step in directed cell migration is the recruitment of cytoskeletal regulatory and signaling proteins to the leading edge of the cell. One protein localized to the leading edge of a migrating astrocyte is beta-catenin. Using an in vitro wound-healing assay, we show that the localization of beta-catenin to the leading edge is dependent upon new protein synthesis at the time of wounding. We examined the mRNA encoding beta-catenin for potential regulatory elements and identified a conserved cytoplasmic polyadenylation element in the 3'-untranslated region (UTR). We now show that the CPE-binding protein (CPEB1) is expressed in astrocytes and that translation of beta-catenin mRNA is regulated by CPEB1. Further, expression of a mutant CPEB1 protein in astrocytes not only blocks beta-catenin protein localization, it also inhibits cell migration. These findings demonstrate a role for CPEB1-mediated protein synthesis in the localization of beta-catenin protein to the leading edge of migrating astrocytes and in regulating directed cell motility.