Epigenome reprogramming through H3K27 and H3K4 trimethylation as a resistance mechanism to DNA methylation inhibition in BRAFV600E-mutated colorectal cancer.

PURPOSE: BRAFV600E-mutated colorectal cancer (CRC) exhibits a strong correlation with DNA hypermethylation suggesting this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E CRC in vivo. EXPERIMENTAL DESIGN: We randomized and treated mice implanted with patient-derived tumor xenografts harboring BRAFV600E mutation with control, 5-azacitidine, vemurafenib (BRAF inhibitor), or the combination. Comprehensive epigenomic profiling was conducted on control and 5-azacitidine-treated tumor samples, including DNA methylation, histone modifications, chromatin accessibility, and gene expression. Combinations of epigenetic agents were explored in preclinical BRAFV600E CRC models. RESULTS: A profound reduction of DNA methylation levels upon 5-azacitidine treatment was confirmed, however, transcriptional repression was not relieved. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon 5-azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around these hypomethylated regions suggesting the involvement of polycomb repressive complex (PRC) activity around the genome with loss of DNA methylation, therefore maintaining the repression of key tumor suppressor genes. Combined inhibition of PRC activity through EZH2 inhibitor with 5-azacitidine treatment additively improved efficacies in BRAFV600E CRC cells. CONCLUSIONS: In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E CRC, and simultaneous blockade of DNMT and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated CRC.

Therapeutic targeting of Syndecan-1 axis overcomes acquired resistance to KRAS-targeted therapy in gastrointestinal cancers.

The therapeutic benefit of recently developed mutant KRAS (mKRAS) inhibitors has been limited by the rapid onset of resistance. Here, we aimed to delineate the mechanisms underlying acquired resistance to mKRAS inhibition and identify actionable targets for overcoming this clinical challenge. Previously, we identified Syndecan-1 (SDC1) as a key effector for pancreatic cancer progression whose surface expression is driven by mKRAS. By leveraging both pancreatic and colorectal cancer models, we found that surface SDC1 expression was initially diminished upon mKRAS inhibition, but recovered in tumor cells that bypass mKRAS dependency. Functional studies showed that these tumors depended on SDC1 for survival, further establishing SDC1 as a driver for the acquired resistance to mKRAS inhibition. Mechanistically, we revealed that the YAP1-SDC1 axis was the major driving force for bypassing mKRAS dependency to sustain nutrient salvage machinery and tumor maintenance. Specifically, YAP1 activation mediated the recovery of SDC1 localization on cell surface that sustained macropinocytosis and enhanced the activation of multiple RTKs, promoting resistance to KRAS-targeted therapy. Overall, our study has provided the rationale for targeting the YAP-SDC1 axis to overcome resistance to mKRAS inhibition, thereby revealing new therapeutic opportunities for improving the clinical outcome of patients with KRAS-mutated cancers.

Co-targeting SOS1 enhances the antitumor effects of KRASG12C inhibitors by addressing intrinsic and acquired resistance.

Combination approaches are needed to strengthen and extend the clinical response to KRASG12C inhibitors (KRASG12Ci). Here, we assessed the antitumor responses of KRASG12C mutant lung and colorectal cancer models to combination treatment with a SOS1 inhibitor (SOS1i), BI-3406, plus the KRASG12C inhibitor, adagrasib. We found that responses to BI-3406 plus adagrasib were stronger than to adagrasib alone, comparable to adagrasib with SHP2 (SHP2i) or EGFR inhibitors and correlated with stronger suppression of RAS-MAPK signaling. BI-3406 plus adagrasib treatment also delayed the emergence of acquired resistance and elicited antitumor responses from adagrasib-resistant models. Resistance to KRASG12Ci seemed to be driven by upregulation of MRAS activity, which both SOS1i and SHP2i were found to potently inhibit. Knockdown of SHOC2, a MRAS complex partner, partially restored response to KRASG12Ci treatment. These results suggest KRASG12C plus SOS1i to be a promising strategy for treating both KRASG12Ci naive and relapsed KRASG12C-mutant tumors.

Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

Fruit and seed structure in the ANA-grade angiosperms: Ancestral traits and specializations.

PREMISE: The representatives of the ANA-grade angiosperms demonstrate a diverse pattern of morphological characters, but their apocarpous gynoecium (except in Nymphaeaceae), composed of at least partly ascidiate carpels, the four-nucleate and four-celled female gametophyte, and the diploid endosperm (except in Amborella) are inferred to be plesiomorphies. Since the structure of fruits in Austrobaileyales is under-investigated, this research aims to fill this gap in these data, describing the carpological characters of ANA-grade taxa, and potentially illuminating the ancestral fruit and seed types of angiosperms. METHODS: The pericarp and seed coat anatomy was studied with light microscopy. The character optimization was carried out using WinClada software. RESULTS: The fruits of Austrobaileya, Trimenia, Kadsura, and Schisandra are determined to be apocarpous berries of the Schisandra type, with a parenchymatous pericarp and mesotestal (Austrobaileya) or exomesotestal seeds (other genera). Most inferred scenarios of fruit evolution indicate that the apocarpous berry is either the most probable plesiomorphic fruit type of all angiosperms, or that of all angiosperms except Amborellaceae. This inference suggests the early origin of the berry in fruit evolution. The plesiomorphic seed type of angiosperms according to reconstructed scenarios of seed type evolution was either a seed lacking a sclerenchymatous layer or an exotestal seed. CONCLUSIONS: The current research indicates that an apocarpous berry, and not a follicle, is a probable plesiomorphic character of the ANA-grade taxa and of angiosperms as a whole.

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

Antitumor Efficacy of Dual Blockade with Encorafenib + Cetuximab in Combination with Chemotherapy in Human BRAFV600E-Mutant Colorectal Cancer.

PURPOSE: Encorafenib + cetuximab (E+C) is an effective therapeutic option in chemorefractory BRAFV600E metastatic colorectal cancer (mCRC). However, there is a need to improve the efficacy of this molecular-targeted therapy and evaluate regimens suitable for untreated BRAFV600E in patients with mCRC. EXPERIMENTAL DESIGN: We performed a series of in vivo studies using BRAFV600E mCRC tumor xenografts. Mice were randomized to receive 5-fluoruracil (5-FU), irinotecan, or oxaliplatin regimens (FOLFIRI or FOLFOX), (E+C) or the combination. Patients received long-term treatment until disease progression, with deescalation strategies used to mimic maintenance therapy. Transcriptomic changes after progression on cytotoxic chemotherapy or targeted therapy were assessed. RESULTS: Antitumor activity of either FOLFIRI or E+C was better as first-line treatment as compared with second-line, with partial cross-resistance seen between a cytotoxic regimen and targeted therapy with an average 62% loss of efficacy for FOLFIRI after E+C and a 45% loss of efficacy of E+C after FOLFIRI (P < 0.001 for both). FOLFIRI-treated models had upregulation of epithelial-mesenchymal transition (EMT) and MAPK pathway activation, where E+C treated models had suppressed MAPK signaling. In contrast, with chemotherapy with E+C, EMT and MAPK signaling remained suppressed. FOLFOX or FOLFIRI, each in combination with E+C, were the most active first-line treatments as compared with E+C or to chemotherapy alone. Furthermore, FOLFOX in combination with E+C as first-line induction therapy, followed by E+C ± 5-FU as maintenance therapy, was the most effective strategy for long-term disease control. CONCLUSIONS: These results support the combination of cytotoxic chemotherapy and molecular-targeted therapy as a promising therapeutic approach in the first-line treatment of BRAFV600E mCRC.

Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo.

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.

Combined KRASG12C and SOS1 inhibition enhances and extends the anti-tumor response in KRASG12C-driven cancers by addressing intrinsic and acquired resistance.

Efforts to improve the anti-tumor response to KRASG12C targeted therapy have benefited from leveraging combination approaches. Here, we compare the anti-tumor response induced by the SOS1-KRAS interaction inhibitor, BI-3406, combined with a KRASG12C inhibitor (KRASG12Ci) to those induced by KRASG12Ci alone or combined with SHP2 or EGFR inhibitors. In lung cancer and colorectal cancer (CRC) models, BI-3406 plus KRASG12Ci induces an anti-tumor response stronger than that observed with KRASG12Ci alone and comparable to those by the other combinations. This enhanced anti-tumor response is associated with a stronger and extended suppression of RAS-MAPK signaling. Importantly, BI-3406 plus KRASG12Ci treatment delays the emergence of acquired adagrasib resistance in both CRC and lung cancer models and is associated with re-establishment of anti-proliferative activity in KRASG12Ci-resistant CRC models. Our findings position KRASG12C plus SOS1 inhibition therapy as a promising strategy for treating both KRASG12C-mutated tumors as well as for addressing acquired resistance to KRASG12Ci.

Resistance Mechanisms to Anti-Epidermal Growth Factor Receptor Therapy in RAS/RAF Wild-Type Colorectal Cancer Vary by Regimen and Line of Therapy.

PURPOSE: Acquired resistance to anti-epidermal growth factor receptor (EGFR) inhibitor (EGFRi) therapy in colorectal cancer (CRC) has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR, among other MAPK-pathway members. However, this was primarily on the basis of single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients. METHODS: We analyzed paired plasma samples from patients with RAS/BRAF/EGFR wild-type metastatic CRC enrolled in three large randomized trials evaluating EGFRi in the first line in combination with chemotherapy and as a single agent in third line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, infusional fluorouracil, leucovorin, and oxaliplatin, and SN38 were developed, and transcriptional changes profiled. RESULTS: Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared with those treated in combination with cytotoxic chemotherapy (9%). Furthermore, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition. By contrast, commonly acquired resistance alterations in the MAPK pathway do not affect sensitivity to cytotoxic chemotherapy. CONCLUSION: These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development. SIGNIFICANCE: This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.

KRASG12C-independent feedback activation of wild-type RAS constrains KRASG12C inhibitor efficacy.

Although KRAS has long been considered undruggable, direct KRASG12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRASG12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRASG12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS.

Targeting XPO1-Dependent Nuclear Export in Cancer.

Nucleocytoplasmic transport of macromolecules is tightly regulated in eukaryotic cells. XPO1 is a transport factor responsible for the nuclear export of several hundred protein and RNA substrates. Elevated levels of XPO1 and recurrent mutations have been reported in multiple cancers and linked to advanced disease stage and poor survival. In recent years, several novel small-molecule inhibitors of XPO1 were developed and extensively tested in preclinical cancer models and eventually in clinical trials. In this brief review, we summarize the functions of XPO1, its role in cancer, and the latest results of clinical trials of XPO1 inhibitors.

Immunization with recombinant ORF2 p551 protein protects common marmosets (Callithrix jacchus) against homologous and heterologous hepatitis E virus challenge.

BACKGROUND: Hepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus, CM). METHODS: p551 with consensus sequence corresponding to amino acid residues 110-660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 µg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera. RESULTS: Purified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge. CONCLUSIONS: CMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV.

Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

The Provocative Roles of Platelets in Liver Disease and Cancer.

Both platelets and the liver play important roles in the processes of coagulation and innate immunity. Platelet responses at the site of an injury are rapid; their immediate activation and structural changes minimize the loss of blood. The majority of coagulation proteins are produced by the liver-a multifunctional organ that also plays a critical role in many processes: removal of toxins and metabolism of fats, proteins, carbohydrates, and drugs. Chronic inflammation, trauma, or other causes of irreversible damage to the liver can dysregulate these pathways leading to organ and systemic abnormalities. In some cases, platelet-to-lymphocyte ratios can also be a predictor of disease outcome. An example is cirrhosis, which increases the risk of bleeding and prothrombotic events followed by activation of platelets. Along with a triggered coagulation cascade, the platelets increase the risk of pro-thrombotic events and contribute to cancer progression and metastasis. This progression and the resulting tissue destruction is physiologically comparable to a persistent, chronic wound. Various cancers, including colorectal cancer, have been associated with increased thrombocytosis, platelet activation, platelet-storage granule release, and thrombosis; anti-platelet agents can reduce cancer risk and progression. However, in cancer patients with pre-existing liver disease who are undergoing chemotherapy, the risk of thrombotic events becomes challenging to manage due to their inherent risk for bleeding. Chemotherapy, also known to induce damage to the liver, further increases the frequency of thrombotic events. Depending on individual patient risks, these factors acting together can disrupt the fragile balance between pro- and anti-coagulant processes, heightening liver thrombogenesis, and possibly providing a niche for circulating tumor cells to adhere to-thus promoting both liver metastasis and cancer-cell survival following treatment (that is, with minimal residual disease in the liver).

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

PURPOSE: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. EXPERIMENTAL DESIGN: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. RESULTS: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. CONCLUSIONS: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Randomized Trial of Irinotecan and Cetuximab With or Without Vemurafenib in BRAF-Mutant Metastatic Colorectal Cancer (SWOG S1406).

PURPOSE: BRAFV600E mutations are rarely associated with objective responses to the BRAF inhibitor vemurafenib in patients with metastatic colorectal cancer (CRC). Blockade of BRAFV600E by vemurafenib causes feedback upregulation of EGFR, whose signaling activities can be impeded by cetuximab. METHODS: One hundred six patients with BRAFV600E-mutated metastatic CRC previously treated with one or two regimens were randomly assigned to irinotecan and cetuximab with or without vemurafenib (960 mg PO twice daily). RESULTS: Progression-free survival, the primary end point, was improved with the addition of vemurafenib (hazard ratio, 0.50, P = .001). The response rate was 17% versus 4% (P = .05), with a disease control rate of 65% versus 21% (P < .001). A decline in circulating tumor DNA BRAFV600E variant allele frequency was seen in 87% versus 0% of patients (P < .001), with a low incidence of acquired RAS alterations at the time of progression. RNA profiling suggested that treatment benefit did not depend on previously established BRAF subgroups or the consensus molecular subtype. CONCLUSION: Simultaneous inhibition of EGFR and BRAF combined with irinotecan is effective in BRAFV600E-mutated CRC.

YB-1 Phosphorylation at Serine 209 Inhibits Its Nuclear Translocation.

YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that is transported to the nucleus in certain conditions, including DNA-damaging stress, transcription inhibition, and viral infection. In tumors, YB-1 nuclear localization correlates with high aggressiveness, multidrug resistance, and a poor prognosis. It is known that posttranslational modifications can regulate the nuclear translocation of YB-1. In particular, well-studied phosphorylation at serine 102 (S102) activates YB-1 nuclear import. Here, we report that Akt kinase phosphorylates YB-1 in vitro at serine 209 (S209), which is located in the vicinity of the YB-1 nuclear localization signal. Using phosphomimetic substitutions, we showed that S209 phosphorylation inhibits YB-1 nuclear translocation and prevents p-S102-mediated YB-1 nuclear import.