ERG activates a stem-like proliferation-differentiation program in prostate epithelial cells with mixed basal-luminal identity.

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in all prostate epithelial cells, proliferation was enriched in a small, stem-like population with mixed-luminal basal identity (called intermediate cells). Through a series of lineage tracing and primary prostate tissue transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that co-express the luminal genes Tmprss2 and Nkx3.1 (called BasalLum) but not in the larger population of classical Krt8+ luminal cells. Upon ERG activation, BasalLum cells give rise to the highly proliferative intermediate state, which subsequently transitions to the larger population of Krt8+ luminal cells characteristic of ERG-positive human cancers. Furthermore, this proliferative population is characterized by an ERG-specific chromatin state enriched for NFkB, AP-1, STAT and NFAT binding, with implications for TF cooperativity. The fact that the proliferative potential of ERG is enriched in a small stem-like population implicates the chromatin context of these cells as a critical variable for unmasking its oncogenic activity.

The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1.

Lineage plasticity is a recognized hallmark of cancer progression that can shape therapy outcomes. The underlying cellular and molecular mechanisms mediating lineage plasticity remain poorly understood. Here, we describe a versatile in vivo platform to identify and interrogate the molecular determinants of neuroendocrine lineage transformation at different stages of prostate cancer progression. Adenocarcinomas reliably develop following orthotopic transplantation of primary mouse prostate organoids acutely engineered with human-relevant driver alterations (e.g., Rb1-/-; Trp53-/-; cMyc+ or Pten-/-; Trp53-/-; cMyc+), but only those with Rb1 deletion progress to ASCL1+ neuroendocrine prostate cancer (NEPC), a highly aggressive, androgen receptor signaling inhibitor (ARSI)-resistant tumor. Importantly, we show this lineage transition requires a native in vivo microenvironment not replicated by conventional organoid culture. By integrating multiplexed immunofluorescence, spatial transcriptomics and PrismSpot to identify cell type-specific spatial gene modules, we reveal that ASCL1+ cells arise from KRT8+ luminal epithelial cells that progressively acquire transcriptional heterogeneity, producing large ASCL1+;KRT8- NEPC clusters. Ascl1 loss in established NEPC results in transient tumor regression followed by recurrence; however, Ascl1 deletion prior to transplantation completely abrogates lineage plasticity, yielding adenocarcinomas with elevated AR expression and marked sensitivity to castration. The dynamic feature of this model reveals the importance of timing of therapies focused on lineage plasticity and offers a platform for identification of additional lineage plasticity drivers.

Combined RAF and MEK Inhibition to Treat Activated Non-V600 BRAF-Altered Advanced Cancers.

BACKGROUND: Cancers with non-V600 BRAF-activating alterations have no matched therapy. Preclinical data suggest that these tumors depend on ERK signaling; however, clinical response to MEK/ERK inhibitors has overall been low. We hypothesized that a narrow therapeutic index, driven by ERK inhibition in healthy (wild-type) tissues, limits the efficacy of these inhibitors. As these mutants signal as activated dimers, we further hypothesized that RAF inhibitors given concurrently would improve the therapeutic index by opposing ERK inhibition in normal tissues and not activate ERK in the already activated tumor. MATERIALS AND METHODS: Using cell lines and patient-derived xenografts, we evaluated the effect of RAF inhibition, alone and in combination with MEK/ERK inhibitors. We then undertook a phase I/II clinical trial of a higher dose of the MEK inhibitor binimetinib combined with the RAF inhibitor encorafenib in patients with advanced cancer with activating non-V600 BRAF alterations. RESULTS: RAF inhibition led to modest inhibition of signaling and growth in activated non-V600 BRAF preclinical models and allowed higher dose of MEK/ERK inhibitors in vivo for more profound tumor regression. Fifteen patients received binimetinib 60 mg twice daily plus encorafenib 450 mg daily (6 gastrointestinal primaries, 6 genitourinary primaries, 3 melanoma, and 2 lung cancer; 7 BRAF mutations and 8 BRAF fusions). Treatment was well tolerated without dose-limiting toxicities. One patient had a confirmed partial response, 8 had stable disease, and 6 had radiographic or clinical progression as best response. On-treatment biopsies revealed incomplete ERK pathway inhibition. CONCLUSION: Combined RAF and MEK inhibition does not sufficiently inhibit activated non-V600 BRAF-mutant tumors in patients.

Blocking the CXCL1-CXCR2 axis enhances the effects of doxorubicin in HCC by remodelling the tumour microenvironment via the NF-κB/IL-1ß/CXCL1 signalling pathway.

Inflammation is a core mechanism for oncogenesis. Chemokines act as important mediators of chronic inflammation and the tumour inflammatory response. However, there is limited information on chemokines in hepatocellular carcinoma (HCC), a disease for which almost all cases are derived from chronic liver inflammation. Here, we explored the protumor effects of CXCL1, a commonly elevated inflammatory chemokine in cirrhosis, in HCC. The protumor role was confirmed in clinical samples from HCC patients. CXCL1 enhanced tumorigenesis in the hepatic inflammatory microenvironment directly by acting on tumour cells and indirectly through promoting the recruitment of macrophages. The increase in the number of macrophages in the tumour microenvironment (TME) promoted tumour cell epithelial-mesenchymal transition (EMT) and significantly increased CXCL1 levels in the TME partly through NF-κB/IL-1ß activation. To investigate the potential therapeutic value of CXCL1 in HCC with an inflammatory background, an antibody blocking CXCL1 was used alone or combined with the chemotherapy agent doxorubicin (DOX), with the goal of reshaping the TME. It has been shown that blocking CXCL1-CXCR2 inhibits tumour progression and reduces macrophage recruitment in the TME. The combination regimen has been shown to synergistically reduce the number of pro-tumour macrophages in the TME and suppress tumour progression. This provides insight into therapeutic strategies for treating HCC patients with high CXCL1 expression.

Hepatocellular carcinoma-derived FOXO1 inhibits tumor progression by suppressing IL-6 secretion from macrophages.

Tumor heterogeneity dominates tumor biological behavior and shapes the tumor microenvironment. However, the mechanisms of tumor genetic features modulate immunity response were not clearly clarified. Tumor associated macrophages (TAMs) exert distinct immune functions in the progression of hepatocellular carcinoma (HCC) based on the inducible phenotype. FOXO family members sense changes in the extracellular or intracellular environment by activating a series of signaling pathways. FOXO1, a transcription factor that a common suppressor in hepatocellular carcinoma, correlated with a better tumor biological behavior in HCC through shaping macrophages anti-tumour response. Here, we found that human HCC tissue microarray (TMA) slides were employed to showed tumor derived FOXO1 negatively related with distribution of protumour macrophages. This phenomenon was confirmed in mouse xenograft model and in vitro. HCC-derived FOXO1 inhibits tumorigenesis not only by targeting tumor cells but also by synchronizing with re-educated macrophages. These effects may be partially dependent on FOXO1 transcriptionally modulates IRF-1/nitrio oxide (NO) axis in exerting effects in macrophages and decreasing IL-6 releasing from macrophages in tumor microenvironment indirectly. This feedback suppressed the progression of HCC by inactivation of IL-6/STAT3 in HCC. It implicates the potential role of FOXO1 in the therapeutic effects for modulating immune response by targeting macrophages.

STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma.

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFß. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.

Molecular Characterization of Acquired Resistance to KRASG12C-EGFR Inhibition in Colorectal Cancer.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. SIGNIFICANCE: Clinical resistance to KRASG12C-EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches. This article is highlighted in the In This Issue feature, p. 1.

ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1.

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Targeting the mTOR Pathway in Hurthle Cell Carcinoma Results in Potent Antitumor Activity.

Hurthle cell carcinomas (HCCs) are refractory to radioactive iodine and unresponsive to chemotherapeutic agents, with a fatality rate that is the highest among all types of thyroid cancer after anaplastic thyroid cancer. Our previous study on the genomic landscape of HCCs identified a high incidence of disruptions of mTOR pathway effectors. Here, we report a detailed analysis of mTOR signaling in cell line and patient-derived xenograft mouse models of HCCs. We show that mTOR signaling is upregulated and that targeting mTOR signaling using mTOR inhibitors suppresses tumor growth in primary tumors and distant metastasis. Mechanistically, ablation of mTOR signaling impaired the expression of p-S6 and cyclin A2, resulting in the decrease of the S phase and blocking of cancer cell proliferation. Strikingly, mTOR inhibitor treatment significantly reduced lung metastatic lesions, with the decreased expression of Snail in xenograft tumors. Our data demonstrate that mTOR pathway blockade represents a novel treatment strategy for HCC.

ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody.

PURPOSE: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. EXPERIMENTAL DESIGN: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. RESULTS: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. CONCLUSIONS: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Prognostic and therapeutic significance of COP9 signalosome subunit CSN5 in prostate cancer.

Chromosome 8q gain is associated with poor clinical outcomes in prostate cancer, but the underlying biological mechanisms remain to be clarified. CSN5, a putative androgen receptor (AR) partner that is located on chromosome 8q, is the key subunit of the COP9 signalosome, which deactivates ubiquitin ligases. Deregulation of CSN5 could affect diverse cellular functions that contribute to tumor development, but there has been no comprehensive study of its function in prostate cancer. The clinical significance of CSN5 amplification/overexpression was evaluated in 16 prostate cancer clinical cohorts. Its oncogenic activity was assessed by genetic and pharmacologic perturbations of CSN5 activity in prostate cancer cell lines. The molecular mechanisms of CSN5 function were assessed, as was the efficacy of the CSN5 inhibitor CSN5i-3 in vitro and in vivo. Finally, the transcription cofactor activity of CSN5 in prostate cancer cells was determined. The prognostic significance of CSN5 amplification and overexpression in prostate cancer was independent of MYC amplification. Inhibition of CSN5 inhibited its oncogenic function by targeting AR signaling, DNA repair, multiple oncogenic pathways, and spliceosome regulation. Furthermore, inhibition of CSN5 repressed metabolic pathways, including oxidative phosphorylation and glycolysis in AR-negative prostate cancer cells. Targeting CSN5 with CSN5i-3 showed potent antitumor activity in vitro and in vivo. Importantly, CSN5i-3 synergizes with PARP inhibitors to inhibit prostate cancer cell growth. CSN5 functions as a transcription cofactor to cooperate with multiple transcription factors in prostate cancer. Inhibiting CSN5 strongly attenuates prostate cancer progression and could enhance PARP inhibition efficacy in the treatment of prostate cancer.

Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis.

FBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and -2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.

Rapid interrogation of cancer cell of origin through CRISPR editing.

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

The role of miRNA125b in the progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common tumours worldwide, and identifying markers related to HCC is an important area of research. As a microRNA (miRNA), miRNA125b (miR-125b) plays an important role in the prediction and prognosis of HCC. In the past 10 years, with increasing research on miR-125b and HCC, the molecular mechanism of its relationship with the development of HCC has been elucidated. MiR-125b inhibits the development of HCC and is highly accurate in predicting HCC and is therefore a valuable predictive marker of HCC. This article summarizes the clinical application of miR-125b in HCC and the potential mechanism of its involvement in the progression of HCC.

Tumor Microenvironment-Derived NRG1 Promotes Antiandrogen Resistance in Prostate Cancer.

Despite the development of second-generation antiandrogens, acquired resistance to hormone therapy remains a major challenge in treating advanced prostate cancer. We find that cancer-associated fibroblasts (CAFs) can promote antiandrogen resistance in mouse models and in prostate organoid cultures. We identify neuregulin 1 (NRG1) in CAF supernatant, which promotes resistance in tumor cells through activation of HER3. Pharmacological blockade of the NRG1/HER3 axis using clinical-grade blocking antibodies re-sensitizes tumors to hormone deprivation in vitro and in vivo. Furthermore, patients with castration-resistant prostate cancer with increased tumor NRG1 activity have an inferior response to second-generation antiandrogen therapy. This work reveals a paracrine mechanism of antiandrogen resistance in prostate cancer amenable to clinical testing using available targeted therapies.

EGFR Blockade Reverts Resistance to KRASG12C Inhibition in Colorectal Cancer.

Most patients with KRAS G12C-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRAS G12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRAS G12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRAS G12C colorectal cancer. SIGNIFICANCE: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079.

Stomatin-like Protein 2 Promotes Tumor Cell Survival by Activating the JAK2-STAT3-PIM1 Pathway, Suggesting a Novel Therapy in CRC.

Despite intensive efforts, a considerable proportion of colorectal cancer (CRC) patients develop local recurrence and distant metastasis. Stomatin-like protein 2 (SLP-2), a member of the highly conserved stomatin superfamily, is upregulated across cancer types. However, the biological and functional roles of SLP-2 remain elusive in CRC. Here, we report that high SLP-2 expression was found in CRC tissues and was linked to tumor progression and tumor cell differentiation. Additionally, high SLP-2 expression correlated with poor overall survival (OS) in CRC patients (p < 0.001). SLP-2 knockout (SLP-2KO), generated by CRISPR/Cas9, reduced cell growth, migration, and invasion; induced apoptosis in CRC cells; and reduced tumor xenograft growth in vivo. A 181-compound library screening showed that SLP-2KO produced resistance to JAK2 inhibitors (NVP-BSK805 and TG-101348) and a PIM1 inhibitor (SGI-1776), revealing that the JAK2-STAT3-PIM1 oncogenic pathway was potentially controlled by SLP-2 in CRC. In vitro and in vivo, TG-101348 combined with SGI-1776 was synergistic in CRC (combination index [CI] < 1). Overall, our findings suggest that SLP-2 controls the JAK2-STAT3-PIM1 oncogenic pathway, offering a rationale for a novel therapeutic strategy with combined SGI-1776 and TG-101348 in CRC. Additionally, SLP-2 may be a prognostic marker and biomarker for sensitivity to JAK2 and PIM1 inhibitors.

ARID1A determines luminal identity and therapeutic response in estrogen-receptor-positive breast cancer.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER+) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER+ breast cancer. An epigenome CRISPR-CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the ER degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER-FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER+ breast cancer.

Response to Anti-EGFR Therapy in Patients with BRAF non-V600-Mutant Metastatic Colorectal Cancer.

PURPOSE: While mutations in BRAF in metastatic colorectal cancer (mCRC) most commonly occur at the V600 amino acid, with the advent of next-generation sequencing, non-V600 BRAF mutations are increasingly identified in clinical practice. It is unclear whether these mutants, like BRAF V600E, confer resistance to anti-EGFR therapy. EXPERIMENTAL DESIGN: We conducted a multicenter pooled analysis of consecutive patients with non-V600 BRAF-mutated mCRCs identified between 2010 and 2017. Non-V600 BRAF mutations were divided into functional classes based on signaling mechanism and kinase activity: activating and RAS-independent (class 2) or kinase-impaired and RAS-dependent (class 3). RESULTS: Forty patients with oncogenic non-V600 BRAF-mutant mCRC received anti-EGFR antibody treatment [n = 12 (30%) class 2 and n = 28 (70%) class 3]. No significant differences in clinical characteristics were observed by mutation class. In contrast, while only 1 of 12 patients with class 2 BRAF mCRC responded, 14 of 28 patients with class 3 BRAF responded to anti-EGFR therapy (response rate, 8% and 50%, respectively, P = 0.02). Specifically, in first- or second-line, 1 of 6 (17%) patients with class 2 and 7 of 9 (78%) patients with class 3 BRAF mutants responded (P = 0.04). In third- or later-line, none of 6 patients with class 2 and 7 of 19 (37%) patients with class 3 BRAF mutants responded (P = 0.14). CONCLUSIONS: Response to EGFR antibody treatment in mCRCs with class 2 BRAF mutants is rare, while a large portion of CRCs with class 3 BRAF mutants respond. Patients with colorectal cancer with class 3 BRAF mutations should be considered for anti-EGFR antibody treatment.See related commentary by Fontana and Valeri, p. 6896.

V211D Mutation in MEK1 Causes Resistance to MEK Inhibitors in Colon Cancer.

We report the emergence of the novel MEK1 V211D gatekeeper mutation in a patient with BRAF K601E colon cancer treated with the allosteric MEK inhibitor binimetinib and the anti-EGFR antibody panitumumab. The MEK1 V211D mutation concurrently occurs in the same cell with BRAF K601E and leads to RAF-independent activity but remains regulated by RAF. The V211D mutation causes resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutant exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Thus, this mutation serves as a general resistance mutation for current MEK inhibitors; however, it is sensitive to a newly reported ATP-competitive MEK inhibitor, which therefore could be used to overcome drug resistance. SIGNIFICANCE: We report a resistance mechanism to allosteric MEK inhibitors in the clinic. A MEK1 V211D mutation developed in a patient with BRAF K601E colon cancer on MEK and EGFR inhibitors. This mutant increases the catalytic activity of MEK1 and reduces its affinity for binimetinib, but remains sensitive to ATP-competitive MEK inhibitors.This article is highlighted in the In This Issue feature, p. 1143.