Effect of an online educational module incorporating real-time feedback on accuracy of polyp sizing in trainees: a randomized controlled trial.

BACKGROUND: Although polyp size dictates surveillance intervals, endoscopists often estimate polyp size inaccurately. We hypothesized that an intervention providing didactic instruction and real-time feedback could significantly improve polyp size classification. METHODS: We conducted a multicenter randomized controlled trial to evaluate the impact of different components of an online educational module on polyp sizing. Participants were randomized to control (no video, no feedback), video only, feedback only, or video + feedback. The primary outcome was accuracy of polyp size classification into clinically relevant categories (diminutive [1-5mm], small [6-9mm], large [≥10mm]). Secondary outcomes included accuracy of exact polyp size (inmm), learning curves, and directionality of inaccuracy (over- vs. underestimation). RESULTS: 36 trainees from five training programs provided 1360 polyp size assessments. The feedback only (80.1%, P=0.01) and video + feedback (78.9%, P=0.02) groups had higher accuracy of polyp size classification compared with controls (71.6%). There was no significant difference in accuracy between the video only group (74.4%) and controls (P=0.42). Groups receiving feedback had higher accuracy of exact polyp size (inmm) and higher peak learning curves. Polyps were more likely to be overestimated than underestimated, and 29.3% of size inaccuracies impacted recommended surveillance intervals. CONCLUSIONS: Our online educational module significantly improved polyp size classification. Real-time feedback appeared to be a critical component in improving accuracy. This scalable and no-cost educational module could significantly decrease under- and overutilization of colonoscopy, improving patient outcomes while increasing colonoscopy access.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Optimizing Endoscopy Procedure Documentation Improves Guideline-Adherent Care in Upper Gastrointestinal Bleeding.

BACKGROUND AND AIMS: Upper GI bleeding (UGIB) is a common indication for inpatient esophagogastroduodenoscopy (EGD). Guideline adherence improves post-EGD care, including appropriate medication dosing/duration and follow-up procedures that reduce UGIB-related morbidity. We aimed to optimize and standardize post-EGD documentation to improve process and clinical outcomes in UGIB-related care. METHODS: We performed a prospective quality improvement study of inpatient UGIB endoscopies at an academic tertiary referral center during 6/2019-7/2021. Guidelines were used to develop etiology/severity-specific electronic health record note templates. Participants (39 faculty/15 trainees) completed 10-min training in template content/use. We collected pre/post-intervention process data on "Minimal Standard Report" (MSR) documentation including patient disposition, diet, and medications. We also recorded documentation of re-bleed precautions and follow-up procedures. Study outcomes included guideline-based medication prescriptions, ordering of follow-up EGD, and post-discharge re-bleeding. Pre/post-intervention analysis was performed using chi-square tests. RESULTS: From a pre-intervention baseline of 199 patients to 459 patients post-intervention, compliance improved with inpatient PPI (53.4-77.9%, p < 0.001) and discharge PPI (31.3-61.0%, p < 0.001) prescriptions. There was improvement in MSR completion (28.6-42.5%, p < 0.001). Compliance improved with octreotide prescriptions (75.0-93.6%, p = 0.002) and follow-up EGD order (61.3-87.1%, p < 0.001). There was no change in post-discharge re-bleeding. 82.6% of cases used templates. CONCLUSIONS: Our project leveraged endoscopy software to standardize documentation, resulting in improved clinical care behavior and efficiency. Our intervention required low burden of maintenance, and sustainability with high utilization over 9 months. Similar endoscopy templates can be applied to other health systems and procedures to improve care.

Associations of Iron Sucrose and Intradialytic Blood Pressure.

RATIONALE & OBJECTIVE: Intradialytic hypotension and intradialytic hypertension are associated with morbidity and mortality in hemodialysis (HD). Many factors can contribute to intra-HD blood pressure (BP) changes, such as drugs with vasoactive properties that can destabilize an already tenuous BP. Intravenous iron sucrose is commonly administered to correct iron deficiency; however, its reported associations with altered hemodynamics have not been consistent. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 950 outpatients receiving maintenance HD. EXPOSURE: Iron sucrose administered during HD. OUTCOME: Intradialytic hypotension, intradialytic hypertension, systolic blood pressure parameters. ANALYTICAL APPROACH: Unadjusted and adjusted Poisson and linear repeated measures regression models. RESULTS: The mean age of patients included in the study was 53±22 years, 43% were female, and 38% were Black. Mean pre-HD SBP was 152±26 (SD) mm Hg. At baseline, the patients who received higher doses of iron sucrose tended to have diabetes, have longer HD sessions, and have a higher frequency of erythropoiesis-stimulating agent use, compared with those who did not receive iron sucrose. In adjusted models, higher doses of iron sucrose were associated with an 11% lower rate of intradialytic hypotension (incidence rate ratio [IRR] for iron sucrose≥100mg vs 0 mg, 0.89 [95% CI, 0.85-0.94]). In adjusted analyses, the administration of higher doses of iron sucrose during HD was associated with intradialytic hypertension (IRR for iron sucrose≥100mg vs 0 mg, 1.07 [95% CI, 1.04-1.10]). LIMITATIONS: Nonavailability of the precise iron sucrose formulation (volume), laboratory data for each HD session, and outpatient medications. Objective measures of volume status, home medications, and symptom data were not recorded in this study. CONCLUSIONS: We observed an independent association of intravenous iron sucrose administration during HD with a lower risk of intradialytic hypotension and higher risk of intradialytic hypertension. Future studies to better understand the mechanisms underlying these associations are warranted. PLAIN-LANGUAGE SUMMARY: Intradialytic hypotension and intradialytic hypertension are common among patients on hemodialysis, and they are associated with morbidity and mortality. Although many factors may contribute to these risks, medications administered during hemodialysis play an important role. We studied the significance of the intravenous iron sucrose used to treat iron deficiency and the impact it may have on blood pressure during dialysis. In our study of 950 outpatient hemodialysis patients, we observed that administration of iron sucrose was associated with higher systolic blood pressure (during and after hemodialysis sessions) as well as a lower risk of intradialytic hypotension. We also observed that higher doses of iron sucrose are associated with the development of intradialytic hypertension.

Spectrum of Advanced Colorectal Neoplasia and Anticipated Yield of Average-Risk Screening in Veterans Under Age 50.

The incidence of early onset colorectal cancer, or colorectal cancer (CRC) diagnosed before age 50, is increasing.1 In response, multiple societal guidelines in the United States now recommend initiating CRC screening at age 45 in average-risk individuals (ie, those without high-risk clinical characteristics, such as bleeding, or iron deficiency anemia), inflammatory bowel disease, or family history of colorectal neoplasia.2 The Veterans Health Administration (VHA) is the largest integrated health system in the United States and is contending with how best to expand CRC screening access to this younger population in the setting of limited colonoscopy resources. Understanding the rate and anatomic location of colorectal neoplasia in Veterans younger than age 50 can inform the expected yield of different screening modalities. Prior work has shown that individuals undergoing colonoscopy for low-risk diagnostic indications have equivalent risk of colorectal neoplasia as those undergoing average-risk screening.3 This study and a recent meta-analysis4 reported that 3.6% (95% confidence interval, 1.9%-6.7%) to 3.7% (95% confidence interval, 3.0%-4.7%) of average-risk individuals age 45-49 have advanced colorectal neoplasia (ACN), defined as an advanced polyp or carcinoma; however, data specific to the VHA population are lacking.

The rising tide of early-onset colorectal cancer: a comprehensive review of epidemiology, clinical features, biology, risk factors, prevention, and early detection.

The incidence of early-onset colorectal cnacer-ie, colorectal cancer diagnosed in patients under the age of 50 years- has been increasing around the world. This Series paper provides a comprehensive review on the topic of early-onset colorectal cancer, including examining the epidemiology of early-onset colorectal cancer around the world, clinical and pathological features, genetic and epigenetic landscapes, and emerging data on the clinical risk factors associated with this malignancy. Evidence-based approaches to prevention and early detection are also presented.

Trends in the Incidence of Early-Onset Colorectal Adenocarcinoma Among Black and White US Residents Aged 40 to 49 Years, 2000-2017.

Importance: Early-onset colorectal cancer incidence rates are rising faster in White individuals than Black individuals. However, prior National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) racial stratification analyses used smaller SEER 13 databases, combined patients under age 50 years, did not stratify by sex, and did not focus on adenocarcinoma histologic subtypes (screening target). Objective: To perform a race- and sex-stratified adenocarcinoma incidence rate analysis in individuals aged 40 to 49 years using larger SEER 18 databases with expanded race data to better understand the colorectal cancer burden in those at or approaching screening age. Design, Setting, and Participants: This cross-sectional study used 2000 to 2017 SEER 18 annual age-adjusted colorectal cancer incidence rates stratified by anatomic subsite (colon or rectum), adenocarcinoma histology, race (non-Hispanic Black or non-Hispanic White), and sex for individuals aged 40 to 49 years, and yearly annual percent change (APC) incidence rates were calculated. Annual rate ratios (ARRs) between subgroups were determined. Statistical analysis was performed from January to March 2021. Main Outcomes and Measurements: Early-onset colorectal cancer incidence rates, APCs, and ARRs. Results: In this study, a total of 46 728 colorectal cancer cases were identified in 45 429 patients aged 40 to 49 years from 2000 to 2017. Among the 45 429 patients included in this study, 6480 (14.2%) were Black and 27 426 (60.4%) were White; the mean (SD) age was 45.5 (2.8) years. Among White individuals aged 40 to 49 years, colorectal adenocarcinoma incidence rates increased from 19.6 per 100 000 person-years in 2000 to 25.2 per 100 000 person-years in 2017 (APC, 1.6; 95% CI, 1.3 to 1.9). Among Black individuals aged 40 to 49 years, colorectal adenocarcinoma incidence rates were not significantly changed (26.4 per 100 000 person-years in 2000 and 25.8 per 100 000 person-years in 2017 [APC, -0.03; 95% CI, -0.5 to 0.5]). There were no significant differences in ARRs of absolute colorectal incidence rates between White and Black individuals from 2014 to 2017. Rectal-only absolute adenocarcinoma incidence rates in Black and White individuals remained similar from 2000 to 2008 but significantly diverged in 2009. As of 2017, rectal absolute incidence rates were 39% higher among White individuals than among Black individuals with increasing APC (APC, 2.2; 95% CI, 1.6 to 2.8) whereas rectal adenocarcinoma incidence rates among Black individuals were decreasing, although the APC was not statistically significant (APC, -1.4; 95% CI, -2.6 to 0.1). Absolute colonic adenocarcinoma incidence rates remained higher in Black individuals. The study subgroups with the largest divergence in APCs were rectal adenocarcinoma in White vs Black women (APC of 2.2 [95% CI, 1.6 to 2.8] vs APC of -1.7 [95% CI, -3.6 to 0.3], respectively). Conclusions and Relevance: This study found that colorectal adenocarcinoma incidence rates in people aged 40 to 49 years were increasing among White individuals but stabilized among Black individuals with absolute incidence rates becoming equivalent. Absolute rectal adenocarcinoma incidence rates were 39% lower in Black individuals with a widening disparity in rectal cancer between White and Black women. Possible contributors include introduction of a screening threshold of age 45 years in Black individuals in 2008. Although the average-risk screening age has now shifted to age 45 years in all racial groups, these data can help motivate real-world implementation of guidelines to maximize screening rates that have historically been suboptimal in younger individuals.

Active DNA demethylation-The epigenetic gatekeeper of development, immunity, and cancer.

DNA methylation is a critical process in the regulation of gene expression with dramatic effects in development and continually expanding roles in oncogenesis. 5-Methylcytosine was once considered to be an inherited and stably repressive epigenetic mark, which can be only removed by passive dilution during multiple rounds of DNA replication. However, in the past two decades, physiologically controlled DNA demethylation and deamination processes have been identified, thereby revealing the function of cytosine methylation as a highly regulated and complex state-not simply a static, inherited signature or binary on-off switch. Alongside these fundamental discoveries, clinical studies over the past decade have revealed the dramatic consequences of aberrant DNA demethylation. In this review we discuss DNA demethylation and deamination in the context of 5-methylcytosine as critical processes for physiological and physiopathological transitions within three states-development, immune maturation, and oncogenic transformation; and we describe the expanding role of DNA demethylating drugs as therapeutic agents in cancer.

Randomized Controlled Trial of Personalized Colorectal Cancer Risk Assessment vs Education to Promote Screening Uptake.

INTRODUCTION: Risk stratification has been proposed as a strategy to improve participation in colorectal cancer (CRC) screening, but evidence is lacking. We performed a randomized controlled trial of risk stratification using the National Cancer Institute's Colorectal Cancer Risk Assessment Tool (CCRAT) on screening intent and completion. METHODS: A total of 230 primary care patients eligible for first-time CRC screening were randomized to risk assessment via CCRAT or education control. Follow-up of screening intent and completion was performed by record review and phone at 6 and 12 months. We analyzed change in intent after intervention, time to screening, overall screening completion rates, and screening completion by CCRAT risk score tertile. RESULTS: Of the patients, 61.7% of patients were aged <60 years, 58.7% female, and 94.3% with college or higher education. Time to screening did not differ between arms (hazard ratio 0.78 [95% confidence interval (CI) 0.52-1.18], P = 0.24). At 12 months, screening completion was 38.6% with CCRAT vs 44.0% with education (odds ratio [OR] 0.80 [95% CI 0.47-1.37], P = 0.41). Changes in screening intent did not differ between the risk assessment and education arms (precontemplation to contemplation: OR 1.52 [95% CI 0.81-2.86], P = 0.19; contemplation to precontemplation: OR 1.93 [95% CI 0.45-8.34], P = 0.38). There were higher screening completion rates at 12 months in the top CCRAT risk tertile (52.6%) vs the bottom (32.4%) and middle (31.6%) tertiles (P = 0.10). DISCUSSION: CCRAT risk assessment did not increase screening participation or intent. Risk stratification might motivate persons classified as higher CRC risk to complete screening, but unintentionally discourage screening among persons not identified as higher risk.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Modification of the base excision repair enzyme MBD4 by the small ubiquitin-like molecule SUMO1.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Thymine DNA glycosylase as a novel target for melanoma.

Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

Chromosome instability in tumor cells due to defects in Aurora B mediated error correction at kinetochores.

We characterized a panel of cancer cells and found that they exhibited chromosome instability (CIN) that was associated with high frequencies of aberrant kinetochore:microtubule attachments. Failure to resolve these defective attachments before anaphase onset can lead to missegregation of chromosomes. Aurora B kinase is concentrated at the inner centromere where it contributes to multiple kinetochore functions, one of which is in error-correction. Analysis of several CIN cell lines showed that many aspects of Aurora B kinase functions were normal. Furthermore, the amount and activity of Aurora B kinase was not reduced at the kinetochores of CIN cells that were examined. However, phosphorylation of a centromeric biosensor for Aurora B in OVCAR10, MCF7 and U2OS cells was consistently reduced relative to non CIN cells. This suggested a localized problem with Aurora B's ability to phosphorylate substrates important for error correction. This possibility was supported by our ability to improve error correction and reduce the frequency of lagging chromosome in CIN cells by directing endogenous Aurora B to the region of centromere that was tested by the biosensor. Our studies suggest that the kinetochores of CIN cells have a defect that limits accessibility of Aurora B to substrates that are important for error-correction.

Solitary Extrahepatic Intramuscular Metastasis from Cryptogenic Hepatocellular Carcinoma.

Although hepatocellular carcinoma (HCC) recurrence after curative resection is not uncommon, it primarily recurs in the liver prior to metastatic progression. We report a case of resected pT2N0 cryptogenic HCC that recurred in the superior paracervical musculature without evident intrahepatic recurrence. The patient also developed cervical spine instability requiring urgent neurosurgery. Cryptogenic HCC is thought to arise from non-alcoholic fatty liver disease even without cirrhosis. Unfortunately, it also portends a worse prognosis compared to HCC of other etiologies. This highlights the aggressive behavior of cryptogenic HCC, which warrants further research as non-alcoholic fatty liver disease becomes increasingly common.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Methotrexate sensitizes drug-resistant metastatic melanoma cells to BRAF V600E inhibitors dabrafenib and encorafenib.

Acquired resistance of metastatic melanoma (MM) tumors to BRAF V600E inhibitors (BRAFi's) is commonplace in the clinic. Habitual relapse of patients contributes to <20% 5-year survival rates in MM. We previously identified serine synthesis as a critical detrminant of late-stage cancer cell resistance to BRAFi's. Pre-treatment with DNA damaging agent gemcitabine (a nucleoside analog) re-sensitized drug-resistant cancer cells to BRAFi's dabrafenib and vemurafenib. Importantly, the combination treatments were effective against BRAF wild type cancer cells potentially expanding the clinical reach of BRAFi's. In this study, we identify the antifolate methotrexate (MTX) as a sensitizer of acquired- and intrinsically-resistant MM cells to BRAFi's dabrafenib and encorafenib. We identify a novel, positive correlation between dabrafenib treatments and repair delay of MTX induced single-strand DNA (ssDNA) breaks. Cells arrest in G1 phase following simultaneous MTX + dabrafenib treatments and eventually die via apoptosis. Importantly, we identify RAS codon 12 activating mutations as prognostic markers for MTX + BRAFi treatment efficacy. We describe a method of killing drug-resistant MM cells that if translated has the potential to improve MM patient survival.

Identification of the Serine Biosynthesis Pathway as a Critical Component of BRAF Inhibitor Resistance of Melanoma, Pancreatic, and Non-Small Cell Lung Cancer Cells.

Metastatic melanoma cells commonly acquire resistance to BRAF V600E inhibitors (BRAFi). In this study, we identified serine biosynthesis as a critical mechanism of resistance. Proteomic assays revealed differential protein expression of serine biosynthetic enzymes PHGDH, PSPH, and PSAT1 following vemurafenib (BRAFi) treatment in sensitive versus acquired resistant melanoma cells. Ablation of PHGDH via siRNA sensitized acquired resistant cells to vemurafenib. Inhibiting the folate cycle, directly downstream of serine synthesis, with methotrexate also displayed similar sensitization. Using the DNA-damaging drug gemcitabine, we show that gemcitabine pretreatment sensitized resistant melanoma cells to BRAFis vemurafenib and dabrafenib. We extended our findings to BRAF WT tumor cell lines that are intrinsically resistant to vemurafenib and dabrafenib. Pretreatment of pancreatic cancer and non-small cell lung cancer cell lines with sublethal doses of 50 and 5 nmol/L of gemcitabine, respectively, enhanced killing by both vemurafenib and dabrafenib. The novel aspects of this study are the direct identification of serine biosynthesis as a critical mechanism of BRAF V600E inhibitor resistance and the first successful example of using gemcitabine + BRAFis in combination to kill previously drug-resistant cancer cells, creating the translational potential of pretreatment with gemcitabine prior to BRAFi treatment of tumor cells to reverse resistance within the mutational profile and the WT. Mol Cancer Ther; 16(8); 1596-609. ©2017 AACR.