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Ravi G Mirpuri,1 Jereme Brammeier,2 Hamilton Chen,2 Frank PK Hsu,1,3 Vi K Chiu,4 Eric Y Chang1,2,5 1Department of Physical Medicine and Rehabilitaiton, 2Department of Anesthesiology and Perioperative Care, 3Department of Neurological Surgery, 4Department of Medicine, 5Reeve-Irvine Research Center for Spinal Cord Injury, University of California Irvine, Irvine, CA, USA Objective: Hereditary multiple osteochondromas (HMO) usually presents with neoplastic lesions throughout the skeletal system. These lesions frequently cause chronic pain and are conventionally treated with surgical resection and medication. In cases where conventional treatments have failed, spinal cord stimulation (SCS) could be considered as a potential option for pain relief. The objective of this case was to determine if SCS may have a role in treating pain secondary to neoplastic lesions from HMO. Case presentation: We report a 65-year-old female who previously received both surgical and pharmacological interventions for treating HMO neoplastic pain in the lumbar, pelvis, femur, and tibial regions. These interventions either failed to offer significant pain relief or caused excessive lethargy. A SCS trial was then offered with a dual 16-contact lead trial leading to 70%–80% improvement in pain from baseline and 85% reduction in oxycodone IR intake. This was followed by permanent implantation of two 2×8 contact paddle leads (T7–T8 and T9–T10 interspaces). After 8-week follow-up, settings were further optimized resulting in an additional 30% improvement in pain compared to last visit. At 6-month follow-up, the patient reported continued pain relief. Conclusion: This case demonstrates the first successful use of SCS to treat both HMO and nonmalignant neoplastic-related pain. The patient reported pain improvement from baseline, reduced pain medication requirements, and subjective improvement in quality of life. Additionally, this case demonstrates the potential advantage of trialing multiple painful areas with a 16-contact lead in order to avoid multiple trials and placement. Keywords: Exostoses, bone neoplasms/pathology, hereditary exostoses/multiple, back pain/therapy, pain management, spinal cord stimulation, treatment outcome, chronic pain, case reports

Purpose: The outcomes of anti-PD-1 agents plus fluoropyrimidine/platinum in frontline advanced gastroesophageal adenocarcinomas (aGEAs) remain poor. We investigated the safety, tolerability, and activity of fluoropyrimidine/oxaliplatin and tislelizumab with the DKK1-neutralizing antibody DKN-01 in aGEAs in a phase IIa open-label study., Patients and Methods: Patients had untreated human epidermal growth factor receptor 2-negative aGEAs, RECIST v1.1 measurable disease, Eastern Cooperative Oncology Group (ECOG) performance status 0-1, and adequate organ function. Patients received intravenous DKN-01 300 mg once every 2 weeks, tislelizumab 200 mg once every 3 weeks, oxaliplatin 130 mg/m 2 once every 3 weeks, and capecitabine 1,000 mg/m 2 twice daily on days 1-15 of each 21-day cycle. The primary end point was safety and tolerability. Key secondary end points included objective response rate (ORR) by RECISTv1.1, progression-free survival (PFS), and overall survival (OS)., Results: Between September 18, 2020, and April 8, 2021, 25 patients were enrolled. All patients who received at least one dose of DKN-01 were included in the safety analysis. Most patients had gastroesophageal junction tumors, median age was 61 years, 76% were male, and 55% were ECOG of 0. All patients reported at least one treatment-emergent adverse event. The ORR was 73% (95% CI, 49.8 to 89.3), with a disease control rate of 95%. The ORR was 90% (95% CI, 55.5 to 99.7) in the DKK1-high tumor patients and 67% (95% CI, 29.9 to 92.5) in the DKK1-low tumor patients. The median PFS was 11.3 months (95% CI, 5.8 to 12.0) and the 12-month PFS rate was 33%. The median OS was 19.5 months (95% CI, 15.2 to 24.4) with a 12-month OS rate of 76% and an 18-month OS rate of 55%., Conclusion: DKN-01 can be safely combined with frontline fluoropyrimidine/oxaliplatin and tislelizumab and demonstrates encouraging activity independent of PD-L1 expression levels. A randomized phase II trial is ongoing (ClinicalTrials.gov identifier: NCT04363801).

Purpose: Targeted Agent and Profiling Utilization Registry is a phase II basket trial evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancer and genomic alterations known to be drug targets. Results of a cohort of patients with biliary tract cancer (BTC) with ERBB2/3 amplification, overexpression, or mutation treated with pertuzumab plus trastuzumab are reported., Methods: Eligible patients had advanced BTC, measurable disease (RECIST v1.1), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, tumors with ERBB2/3 alterations, and a lack of standard treatment options. Simon's two-stage design was used with a primary end point of disease control (DC), defined as objective response (OR) or stable disease of at least 16+ weeks duration (SD16+) according to RECIST v1.1. Secondary end points included OR, progression-free survival, overall survival, duration of response, duration of stable disease, and safety., Results: Twenty-nine patients were enrolled from February 2017 to January 2022, and all had advanced BTC with an ERBB2/3 alteration. One patient was not evaluable for efficacy. One complete response, eight partial responses, and two SD16+ were observed for DC and OR rates of 40% (90% CI, 27 to 100) and 32% (95% CI, 16 to 52), respectively. The null hypothesis of 15% DC rate was rejected ( P = .0015). Four patients had at least one grade 3 adverse event (AE) or serious AE at least possibly related to treatment: anemia, diarrhea, infusion-related reaction, and fatigue., Conclusion: Pertuzumab plus trastuzumab met prespecified criteria to declare a signal of activity in patients with BTC and ERBB2/3 amplification, overexpression, or mutation.

Purpose: The Targeted Agent and Profiling Utilization Registry (TAPUR) Study is a pragmatic basket trial evaluating antitumor activity of approved targeted agents in patients with advanced cancers harboring potentially actionable genomic alterations. Data from cohorts of patients with high tumor mutational burden (HTMB, defined as ≥9 mutations per megabase) with advanced colorectal cancer (CRC) and other advanced cancers treated with pembrolizumab are reported., Methods: Eligible patients were 18 years and older with measurable tumors and a lack of standard treatment options, an Eastern Cooperative Oncology Group performance status of 0-1, and adequate organ function. The primary end point was disease control (DC), defined as complete or partial response or stable disease (SD) of at least 16-weeks duration. For the CRC cohort, Simon's two-stage design with a null DC rate of 15% versus 35% (power = 0.85; α = .10) was used. Low accruing histology-specific cohorts were collapsed into one histology-pooled (HP) cohort. For the HP cohort, the null hypothesis of a DC rate of 15% was rejected if the lower limit of a one-sided 90% CI was >15%. Secondary end points included objective response (OR), safety, progression-free survival, overall survival, duration of response, and duration of SD., Results: Seventy-seven patients with HTMB with CRC (n = 28) or advanced cancers (n = 49) were treated with pembrolizumab. For the CRC cohort, the DC rate was 31% ( P = .04) and the OR rate was 11%. For the HP cohort, the DC rate was 45% (one-sided 90% CI, 35 to 100) and the OR rate was 26%. The null hypothesis of a 15% DC rate was rejected for both cohorts. Twelve of 77 patients experienced treatment-related grade 3 adverse events (AEs) or serious AEs, including two deaths., Conclusion: Pembrolizumab demonstrated antitumor activity in pretreated patients with advanced cancers and HTMB.

Purpose: The Targeted Agent and Profiling Utilization Registry Study is a phase II basket study evaluating the antitumor activity of commercially available targeted agents in patients with advanced cancers with genomic alterations known to be drug targets. Results of a cohort of patients with solid tumors with ATM mutations treated with nivolumab plus ipilimumab are reported., Methods: Eligible patients had measurable disease (RECIST v.1.1), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. Primary end point was disease control (DC), defined as complete (CR) or partial (PR) response or stable disease (SD) of at least 16 weeks duration (SD16+). Low-accruing histology-specific cohorts with ATM mutations treated with nivolumab plus ipilimumab were collapsed into a single histology-pooled cohort for this analysis. The results were evaluated based on a one-sided exact binomial test with a null DC rate of 15% versus 35% (power = .84; α = .10). Secondary end points were objective response (OR), progression-free survival, overall survival, duration of response, duration of SD, and safety., Results: Twenty-nine patients with 10 tumor types with ATM mutations were enrolled from January 2018 to May 2020. One patient was not evaluable for efficacy. One CR, three PR, and three SD16+ were observed for DC and OR rates of 24% ( P = .13; one-sided 90% CI: 14 to 100) and 14% (95% CI: 4 to 32), respectively. The null hypothesis of 15% DC rate was not rejected. Eleven patients had one treatment-related grade 3 adverse event (AE) or serious AE. There were two treatment-related patient deaths including immune-related encephalitis and respiratory failure., Conclusion: Nivolumab plus ipilimumab did not meet prespecified criteria to declare a signal of activity in patients with solid tumors with ATM mutations.

Purpose: Napabucasin is an investigational, orally administered reactive oxygen species generator bioactivated by intracellular antioxidant NAD(P)H:quinone oxidoreductase 1 that has been evaluated in various solid tumors, including metastatic colorectal cancer (mCRC). Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) is hypothesized to predict response in napabucasin-treated patients with mCRC., Patient and Methods: In the multi-center, open-label, phase III CanStem303C (NCT02753127) study, adults with histologically confirmed mCRC that progressed on first-line fluoropyrimidine plus oxaliplatin ± bevacizumab were randomized to twice-daily napabucasin plus FOLFIRI (napabucasin) or FOLFIRI alone (control). The primary endpoint was overall survival (OS) in the general study population and in patients with pSTAT3-positive tumors (biomarker-positive)., Results: In the general study population (napabucasin, n = 624; control, n = 629), median OS was 14.3 months for napabucasin and 13.8 months for control (hazard ratio [HR], 0.976, one-sided P = .74). Overall, 44% of patients were biomarker-positive (napabucasin, n = 275; control, n = 272). In the biomarker-positive population, median OS was 13.2 months for napabucasin and 12.1 months for control (HR, 0.969; one-sided P > .99). In the control arm, median OS was shorter for biomarker-positive versus biomarker negative patients (12.1 vs. 18.5 months; HR, 1.518; nominal 2-sided P = .0002). The most common treatment-emergent adverse events (TEAEs) were diarrhea (napabucasin, 84.6%; control, 53.9%), nausea (60.5%, 50.5%), vomiting (41.2%, 29.3%), and abdominal pain (41.0%, 25.2%). Grade ≥3 TEAEs occurred in 73.8% of napabucasin-treated and 66.7% of control-treated patients, most commonly diarrhea (21.2%, 7.0%), neutrophil count decreased (13.7%, 19.2%), and neutropenia (13.3%, 15.2%). Safety was similar in biomarker-positive patients., Conclusion: In patients with previously treated mCRC, adding napabucasin to FOLFIRI did not improve OS. Results from the control arm indicate that pSTAT3 is an adverse prognostic factor in mCRC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Genomic testing and targeted use of non-steroidal anti-inflammatory drugs (NSAIDs) may mitigate cancer recurrence risks. This study examines colorectal cancer (CRC) survivors' interest and receptivity to these strategies. Patients diagnosed with stage I-III CRC in 2004-2012 were recruited through the New Mexico Cancer Registry to complete a cancer survivorship experiences survey. We assessed interest in genomic testing, daily aspirin (ASA) and NSAID use, and receptivity to future daily ASA/NSAIDs. Descriptive statistics and multivariable logistic regression models estimated factors associated with genomic testing interest. Receptivity to future ASA/NSAIDs use was estimated for non-users of ASA/NSAIDs. Among CRC survivors (n = 273), 83% endorsed interest in genomic testing, 25% were ASA users and 47% ASA/NSAIDs users. In our final model, genomic testing interest was associated with being uncoupled [OR = 4.11; 95% CI = 1.49-11.35], low income [OR = 0.35, 95% CI: 0.14-0.88], smoking history [OR = 0.35, 95% CI: 0.14-0.90], low [OR: 0.33, 95% CI: 0.07-1.43] and moderate [OR: 0.26, 95% CI: 0.11-0.61] health literacy, and personal CRC risk worry [OR: 2.86, 95% CI: 1.63-5.02, p = 0.0002]. In our final model, ASA use was associated with age [OR: 1.05, 95% CI: 1.01-1.10] and cardiovascular disease history [OR: 2.42, 95% CI: 1.23-4.73, p = 0.010]. Among non-users ASA/NSAIDs, 83% reported receptivity to ASA/NSAIDs to reduce cancer risks, and no significant correlates were identified. The majority of survivors' expressed genomic testing interest and endorsed receptivity toward ASA/NSAIDs use for cancer risk management. Further research to optimize ASA/NSAIDs use guided by genomic testing is warranted., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The aim of the study was to determine safety, antitumor activity, and pharmacodynamic profile of mogamulizumab, an anti-CCR4 monoclonal antibody targeting effector regulatory T cells (Treg) in combination with the checkpoint inhibitor nivolumab in patients with locally advanced or metastatic solid tumors., Patients and Methods: This was a multicenter, dose-finding (phase I), and dose expansion (phase II) study (NCT02705105) in patients with locally advanced or metastatic solid tumors. There were no dose-limiting toxicities in phase I with mogamulizumab 1 mg/kg every week for cycle 1 followed by 1 mg/kg every 2 weeks plus nivolumab 240 mg every 2 weeks intravenously, and cohort expansion occurred at this dose level., Results: All 114 patients treated with mogamulizumab 1 mg/kg plus nivolumab 240 mg in phases I ( n = 4) and II ( n = 110) were assessed for safety and efficacy. Mogamulizumab plus nivolumab showed acceptable safety and tolerability. Objective response rate was 10.5% [95% confidence interval (CI), 5.6-17.7; 3 complete and 9 partial responses]. Disease control rate was 36.8%. Median duration of response was 14.4 months. Median progression-free survival was 2.6 (95% CI, 2.3-3.1) months, and median overall survival was 9.5 (95% CI, 5.9-13.5) months., Conclusions: Combination of mogamulizumab with nivolumab for treatment of patients with locally advanced or metastatic solid tumors did not result in enhanced efficacy. Tolerability of mogamulizumab 1 mg/kg plus nivolumab 240 mg was acceptable., (©2021 The Authors; Published by the American Association for Cancer Research.)

Purpose: Lifestyle factors may have a synergistic effect on health. We evaluated the correlates of poor adherence to a healthy lifestyle among a diverse sample of colorectal cancer (CRC) survivors to inform future lifestyle promotion programs., Methods: Lifestyle questions from a cross-sectional survey were completed by 283 CRC survivors (41% Hispanic, 40% rural, 33% low income). Adherence to recommendations (yes/no) for physical activity, fruit and vegetable servings/day, avoiding tobacco, and healthy weight was summed to create an overall lifestyle quality score. Polytomous logistic regression was used to evaluate correlates of good (reference group), moderate, and poor overall lifestyle quality. Potential correlates included sociodemographic characteristics, cancer-related factors, and indicators of health and well-being., Results: CRC survivors with poor adherence were 2- to 3.4-fold significantly more likely to report multiple comorbidities, poor physical functioning, fatigue, anxiety/depressive symptoms, and poor social participation. In multivariable analyses, poor physical functioning was the only significant correlate of poor adherence to lifestyle recommendations, compared to good adherence [OR (95% CI) 3.4 (1.8-6.4)]. The majority of survivors, 71% and 78%, indicated interest in receiving information on exercise and eating a healthy diet, respectively., Conclusion: Future lifestyle promotion programs for CRC survivors should carefully consider indicators of physical and psychosocial health and well-being, especially poor physical functioning, in the design, recruitment, and implementation of these health programs.

Background: Chemotherapy-induced damage of hematopoietic stem and progenitor cells (HSPC) causes multi-lineage myelosuppression. Trilaciclib is an intravenous CDK4/6 inhibitor in development to proactively preserve HSPC and immune system function during chemotherapy (myelopreservation). Preclinically, trilaciclib transiently maintains HSPC in G1 arrest and protects them from chemotherapy damage, leading to faster hematopoietic recovery and enhanced antitumor immunity., Patients and Methods: This was a phase Ib (open-label, dose-finding) and phase II (randomized, double-blind placebo-controlled) study of the safety, efficacy and PK of trilaciclib in combination with etoposide/carboplatin (E/P) therapy for treatment-naive extensive-stage small-cell lung cancer patients. Patients received trilaciclib or placebo before E/P on days 1-3 of each cycle. Select end points were prespecified to assess the effect of trilaciclib on myelosuppression and antitumor efficacy., Results: A total of 122 patients were enrolled, with 19 patients in part 1 and 75 patients in part 2 receiving study drug. Improvements were seen with trilaciclib in neutrophil, RBC (red blood cell) and lymphocyte measures. Safety on trilaciclib+E/P was improved with fewer ≥G3 adverse events (AEs) in trilaciclib (50%) versus placebo (83.8%), primarily due to less hematological toxicity. No trilaciclib-related ≥G3 AEs occurred. Antitumor efficacy assessment for trilaciclib versus placebo, respectively, showed: ORR (66.7% versus 56.8%, P = 0.3831); median PFS [6.2 versus 5.0 m; hazard ratio (HR) 0.71; P = 0.1695]; and OS (10.9 versus 10.6 m; HR 0.87; P = 0.6107)., Conclusion: Trilaciclib demonstrated an improvement in the patient's tolerability of chemotherapy as shown by myelopreservation across multiple hematopoietic lineages resulting in fewer supportive care interventions and dose reductions, improved safety profile, and no detriment to antitumor efficacy. These data demonstrate strong proof-of-concept for trilaciclib's myelopreservation benefits., Clinical Trail Number: NCT02499770., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Purpose: Improvements in colorectal cancer (CRC) prevention, early detection, and treatment have resulted in substantial gains in survival. However, the health-related quality of life (HRQoL) of CRC survivors often depends on access to supportive care, which differs by survivors' socioeconomic characteristics. The purpose of this study was to investigate the relationship between socioeconomic characteristics and HRQoL in a diverse group of CRC survivors., Methods: We conducted a population-based, cross-sectional study to examine the association between socioeconomic factors (household income, health literacy, and insurance status) and HRQoL domains of pain interference, fatigue, physical function, sleep disturbance, anxiety, and depression. PROMIS® Short Forms v.2.0 were used to assess domains of HRQoL. Linear regression modeling was used to estimate the coefficient representing the average HRQoL domain score and its 95% confidence interval (CI)., Results: Three hundred one CRC survivors participated in the survey. Low-income (≤ $30,000) CRC survivors had, on average, a 4.70-point (95% CI 1.10-8.28) higher pain interference score, a 7.02-point (95% CI 3.27-10.77) higher fatigue score, a 5.13-point (95% CI - 8.56 to - 1.71) lower physical function score, and a 4.44-point (95% 1.40-7.49) higher depression score than CRC survivors with an income ≥ $70,000. Survivors with Medicaid insurance reported significantly greater pain interference and worse physical function than privately insured survivors. Survivors with low health literacy reported significantly greater pain interference compared with survivors with high health literacy., Conclusions: Substantial socioeconomic disparities in HRQoL were observed in this diverse population of CRC survivors., Implications for Cancer Survivors: Designing supportive care interventions to improve HRQoL among low-income and Medicaid-insured CRC survivors is critical for eliminating disparities in CRC outcomes.

Background: Cancer survivors increasingly report financial hardship as a consequence of the high cost of cancer care, yet the financial experience of rural cancer survivors remains largely unstudied. The purpose of this study was to investigate potential rural disparities in the likelihood of financial hardship and nonadherence to surveillance colonoscopy. Methods: Individuals diagnosed with localized or regional colorectal cancer between 2004 and 2012 were ascertained by the population-based New Mexico Tumor Registry. Participants completed a mailed questionnaire or telephone survey about their colorectal cancer survivorship experience, including treatment-related financial hardship and receipt of surveillance colonoscopy. Multivariable logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Results: Compared with urban colorectal cancer survivors ( n = 168), rural colorectal cancer survivors ( n = 109) were slightly older; more likely to be married (65% vs. 59%) and have an annual income <$30,000 (37% vs. 27%); and less likely to be employed (35% vs. 41%), have a college degree (28% vs. 38%), or a high level of health literacy (39% vs. 51%). Rural survivors were twice as likely as urban survivors to report treatment-related financial hardship (OR, 1.86; 95% CI, 1.06-3.28) and nonadherence to surveillance colonoscopy guidelines (OR, 2.28; 95% CI, 1.07-4.85). In addition, financial hardship was independently associated with nonadherence to surveillance colonoscopy (OR, 2.17; 95% CI, 1.01-4.85). Conclusions: Substantial rural disparities in the likelihood of financial hardship and nonadherence to surveillance colonoscopy exist. Impact: Treatment-related financial hardship among rural colorectal cancer survivors may negatively affect adherence to guideline-recommended follow-up care. Cancer Epidemiol Biomarkers Prev; 27(11); 1275-82. ©2018 AACR., (©2018 American Association for Cancer Research.)

Background: Recognition of rare molecular subgroups is a challenge for precision oncology and may lead to tissue-agnostic approval of targeted agents. Here we aimed to comprehensively characterize the clinical, pathological and molecular landscape of RET rearranged metastatic colorectal cancer (mCRC)., Patients and Methods: In this case series, we compared clinical, pathological and molecular characteristics of 24 RET rearranged mCRC patients with those of a control group of 291 patients with RET negative tumors. RET rearranged and RET negative mCRCs were retrieved by systematic literature review and by taking advantage of three screening sources: (i) Ignyta's phase 1/1b study on RXDX-105 (NCT01877811), (ii) cohorts screened at two Italian and one South Korean Institutions and (iii) Foundation Medicine Inc. database. Next-generation sequencing data were analyzed for RET rearranged cases., Results: RET fusions were more frequent in older patients (median age of 66 versus 60 years, P = 0.052), with ECOG PS 1-2 (90% versus 50%, P = 0.02), right-sided (55% versus 32%, P = 0.013), previously unresected primary tumors (58% versus 21%, P < 0.001), RAS and BRAF wild-type (100% versus 40%, P < 0.001) and MSI-high (48% versus 7%, P < 0.001). Notably, 11 (26%) out of 43 patients with right-sided, RAS and BRAF wild-type tumors harbored a RET rearrangement. At a median follow-up of 45.8 months, patients with RET fusion-positive tumors showed a significantly worse OS when compared with RET-negative ones (median OS 14.0 versus 38.0 months, HR: 4.59; 95% CI, 3.64-32.66; P < 0.001). In the multivariable model, RET rearrangements were still associated with shorter OS (HR: 2.97; 95% CI, 1.25-7.07; P = 0.014), while primary tumor location, RAS and BRAF mutations and MSI status were not., Conclusions: Though very rare, RET rearrangements define a new subtype of mCRC that shows poor prognosis with conventional treatments and is therefore worth of a specific management.

The major categories of pancreatic neuroendocrine tumor (PanNET) are well-differentiated NET and poorly differentiated neuroendocrine carcinoma. Sequencing of these tumors has identified multiple important genes in the pathogenesis of PanNETs, such as DAXX/ATRX, MEN1, TP53, RB, and mTOR pathway genes. We identified a case of well-differentiated PanNET with high-grade progression with simultaneous low- and high-grade histologic regions containing variable genomic profiles. We performed tumor microdissection and analyzed both regions using a 409-gene comprehensive cancer panel using next-generation sequencing in addition to immunohistochemical and morphologic studies. The low-grade region showed a change in the DAXX gene as a copy number variant (CNV) deletion. The high-grade region showed CNV deletion changes in the DAXX gene as well as the MEN1 gene. We observed additional mutational changes in the PTEN gene and SMAD4 gene in the high-grade region. Our data support that high-grade progression in PanNETs may be the result of the progressive accumulation of genetic changes (CNVs and point mutational changes) within the body of the tumor. Next generation sequencing may provide pathologists and clinicians with ancillary information to accurately characterize and treat these tumors.

Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer.

There is an unmet clinical need for molecularly directed therapies available for metastatic colorectal cancer. Comprehensive genomic profiling has the potential to identify actionable genomic alterations in colorectal cancer. Through comprehensive genomic profiling we prospectively identified 6 RET fusion kinases, including two novel fusions of CCDC6-RET and NCOA4-RET, in metastatic colorectal cancer (CRC) patients. RET fusion kinases represent a novel class of oncogenic driver in CRC and occurred at a 0.2% frequency without concurrent driver mutations, including KRAS, NRAS, BRAF, PIK3CA or other fusion tyrosine kinases. Multiple RET kinase inhibitors were cytotoxic to RET fusion kinase positive cancer cells and not RET fusion kinase negative CRC cells. The presence of a RET fusion kinase may identify a subset of metastatic CRC patients with a high response rate to RET kinase inhibition. This is the first characterization of RET fusions in CRC patients and highlights the therapeutic significance of prospective comprehensive genomic profiling in advanced CRC.

Background: Clinical therapy for metastatic colorectal cancer (CRC) remains limited, especially when the tumor harbors a KRAS mutation. This study aimed to identify prognostic biomarkers in CRC that are accessible for therapeutic inhibition., Methods: Conditioned media from human CRC epithelial cells and myofibroblasts were screened by cytokine arrays for tumorigenic factors. The protein and mRNA expressions of these factors were determined by immunohistochemistry and gene microarrays in human CRC tissues. Prognostic biomarkers were determined by correlation of mRNA expression to overall survival in stage IV CRC patients. Inhibition of CXCL1 was performed with specific neutralizing antibody and lentiviral shRNAs. Malignant growth was assessed by soft agar growth assays and xenograft tumor growth in immunocompromised mice., Results: CXCL1 was highly secreted by KRAS mutant human CRC cells and myofibroblasts in a complementary adaptive response to serum deprivation. Elevated CXCL1 level promoted anchorage-independent growth of murine fibroblasts and human CRC cells. Inhibition of CXCL1 by neutralizing antibody and specific shRNAs decreased CRC tumor growth. Highly elevated CXCL1 expression significantly correlated with decreased overall survival in stage IV CRC patients (hazard ratio 0.28; 95% CI 0.11-0.72)., Conclusions: High CXCL1 expression is a poor prognostic biomarker in metastatic CRC. CXCL1 inhibition suppressed tumorigenic growth of KRAS mutant CRC cells.

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34(-)Flt3(-) KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34(-)Flt3(-) KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the modulation of angiocrine factors, with Akt-mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs.

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Post-translational modification of Ras proteins includes prenylcysteine-directed carboxyl methylation. Because Ras participates in Erk activation by epidermal growth factor (EGF), we tested whether Ras methylation regulates Erk activation. EGF stimulation of Erk was inhibited by AFC (N-acetyl-S-farnesyl-L-cysteine), an inhibitor of methylation, but not AGC (N-acetyl-S-geranyl-L-cysteine), an inactive analog of AFC. AFC inhibited Ras methylation as well as the activation of pathway enzymes between Ras and Erk but did not inhibit EGF receptor phosphorylation, confirming action at the level of Ras. Transient transfection of human prenylcysteine-directed carboxyl methyltransferase increased EGF-stimulated Erk activation. AFC but not AGC inhibited movement of transiently transfected green fluorescent protein-Ras from the cytosol to the plasma membrane of COS-1 cells and depleted green fluorescent protein-Ras from the plasma membrane in stably transfected Madin-Darby canine kidney cells, suggesting that methylation regulates Erk by ensuring proper membrane localization of Ras. However, when COS-1 cells were transfected with Ras complexed to CD8, plasma membrane localization of Ras was unaffected by AFC, yet EGF-stimulated Erk activation was inhibited by AFC. Thus, Ras methylation appears to regulate Erk activation both through the localization of Ras as well as the propagation of Ras-dependent signals.

Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases., (Copyright The Rockefeller University Press)

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane.

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.

We show that Nras is transiently localized in the Golgi prior to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminated motile, peri-Golgi vesicles, and prolonged BFA treatment blocked PM expression. GFP-Hras colocalized with GFP-Nras, but GFP-Kras4B revealed less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal was required for PM expression, the CAAX motif alone was necessary and sufficient to target proteins to the endomembrane where they were methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif.

The ability of bcl-2 in target cells to block cell-mediated cytotoxicity by allospecific CTL was tested. The blocking effect was variable. Because killing by CTL involves two different pathways, degranulation (perforin plus granzymes) and fas, we examined the effect of bcl-2 on these pathways independently. Bcl-2 in target cells blocked apoptotic cell death induced either by cytotoxic granule extracts or by CTL killing under conditions in which the fas pathway is blocked. On the other hand, bcl-2 had no effect on target cell-killing either by Fas-specific mAb or by CTL capable of killing only via the fas pathway. These data suggest bcl-2 may block apoptotic lysis induced by perforin plus granzymes, but not apoptotic lysis induced via the fas pathway. Thus, any analysis of the effect of bcl-2 on apoptotic cell death in target cells killed by CTL must take into account the relative contributions of the degranulation vs fas pathways.