Combined BRAF, MEK, and heat-shock protein 90 inhibition in advanced BRAF V600-mutant melanoma.

BACKGROUND: Resistance to BRAF and MEK inhibitors in BRAF V600-mutant melanoma is common. Multiple resistance mechanisms involve heat-shock protein 90 (HSP90) clients, and a phase 1 study of vemurafenib with the HSP90 inhibitor XL888 in patients with advanced melanoma showed activity equivalent to that of BRAF and MEK inhibitors. METHODS: Vemurafenib (960 mg orally twice daily) and cobimetinib (60 mg orally once daily for 21 of 28 days) with escalating dose cohorts of XL888 (30, 45, 60, or 90 mg orally twice weekly) was investigated in a phase 1 trial of advanced melanoma, with a modified Ji dose-escalation design. RESULTS: Twenty-five patients were enrolled. After two dose-limiting toxicities (DLTs) (rash and acute kidney injury) in the first cohort, lower doses of vemurafenib (720 mg) and cobimetinib (40 mg) were investigated with the same XL888 doses. Three DLTs (rash) were observed in 12 patients in the XL888 60-mg cohort, and this was determined as the maximum tolerated dose. Objective responses were observed in 19 patients (76%), and the median progression-free survival was 7.6 months, with a 5-year progression-free survival rate of 20%. The median overall survival was 41.7 months, with a 5-year overall survival rate of 37%. Single-cell RNA sequencing was performed on baseline and on-treatment biopsies; treatment was associated with increased immune cell influx (CD4-positive and CD8-positive T cells) and decreased melanoma cells. CONCLUSIONS: Combined vemurafenib and cobimetinib plus XL888 had significant toxicity, requiring frequent dose reductions, which may have contributed to the relatively low progression-free survival despite a high tumor response rate. Given overlapping toxicities, caution must be used when combining HSP90 inhibitors with BRAF and MEK inhibitors.

Evaluation of a Multi-Site Cancer Health Disparities Research Training Program for Underrepresented Undergraduate and Medical Students.

Diversifying the biomedical research workforce is crucial for eliminating cancer health disparities. To address this need, Moffitt Cancer Center and Louisiana State University Health Sciences formed the Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD). A key component of SPIRIT-CHD is the Cancer Research Education Program (CREP), designed to train underrepresented undergraduate and medical students in biomedical science research. The CREP featured an 8-week summer internship with a web-based curriculum, community outreach, and mentored research experiences. Three cohorts (n = 39) completed the CREP. Students were evaluated before and after the internship using the Goal Attainment Scale (GAS), Science Teaching Efficacy Belief Instrument (STEBI), and Research Appraisal Inventory (RAI), modified to assess CREP outcomes. These scales measured students' intentions to pursue cancer research careers, self-efficacy in communicating scientific information, and perceived research abilities. Paired test results showed significant increases (p < 0.001) in scores across the scales (GAS, STEBI, RAI) pre- and post-training. Trainees reported heightened intentions to pursue cancer research careers (GAS; mean increase of 5.3, p < 0.001) and greater self-efficacy in relaying scientific information (STEBI; mean increase of 9.2, p < 0.001). They also showed increased self-confidence in conducting research (RAI; mean increase of 58.2, p < 0.001). These findings demonstrate the program's success in fostering interest in cancer research careers and enhancing research confidence. Results support the development of programs like CREP to positively impact the academic and professional trajectories of underrepresented students, ultimately creating a more diverse and inclusive biomedical research workforce equipped to address health disparities.

Quantifying Cell Heterogeneity and Subpopulations Using Single Cell Metabolomics.

Mass spectrometry (MS) has become an indispensable tool for metabolomics studies. However, due to the lack of applicable experimental platforms, suitable algorithm, software, and quantitative analyses of cell heterogeneity and subpopulations, investigating global metabolomics profiling at the single cell level remains challenging. We combined the Single-probe single cell MS (SCMS) experimental technique with a bioinformatics software package, SinCHet-MS (Single Cell Heterogeneity for Mass Spectrometry), to characterize changes of tumor heterogeneity, quantify cell subpopulations, and prioritize the metabolite biomarkers of each subpopulation. As proof of principle studies, two melanoma cancer cell lines, the primary (WM115; with a lower drug resistance) and the metastatic (WM266-4; with a higher drug resistance), were used as models. Our results indicate that after the treatment of the anticancer drug vemurafenib, a new subpopulation emerged in WM115 cells, while the proportion of the existing subpopulations was changed in the WM266-4 cells. In addition, metabolites for each subpopulation can be prioritized. Combining the SCMS experimental technique with a bioinformatics tool, our label-free approach can be applied to quantitatively study cell heterogeneity, prioritize markers for further investigation, and improve the understanding of cell metabolism in human diseases and response to therapy.

Improving Multi-site Interaction Through Remote Learning Technology: Report from a Training Program to Increase Underrepresented Undergraduate and Medical Students in Health Disparities Research.

Since 2018, we have evaluated the effectiveness of various teaching technologies for training young investigators on translational research in cancer health disparities. The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Moffitt Cancer Center and the Louisiana State University Health Sciences Center. One of the main components of the SPIRIT-CHD is the Cancer Research Education Program (CREP) for training undergraduate and medical students from underrepresented backgrounds. The CREP utilizes a web-based didactic curriculum to engage students at both institutions in biobanking, precision medicine, and cancer health disparities topics. We report experiences from our cross-institutional cancer education program, specifically evaluating the cohorts' satisfaction and learning gains using various communication technologies and instructional approaches. Trainees completed a survey with questions evaluating the curriculum and technology. Trainees reported satisfaction with the flipped classroom model (FCM) content and overall program (mean score = 3.2, SD = 0.79), and would recommend the program to peers. Yet, despite improved program delivery, trainees felt interaction between the two sites (mean score = 1.5, SD = 0.85) and engagement with faculty (mean score = 2.80, SD = 1.14) could be improved. The technology with the highest reported use was e-mail, with a mean score of 4.6 (SD = 0.52). LinkedIn and Twitter had the lowest frequency of use with mean scores at 1.90 (SD = 0.99) and 1.30 (SD = 1.34). Our study highlights the successes and challenges of remote learning using technology to increase interaction and engagement among trainees and faculty in a multi-site cancer research training program.

Development of Malignancy-Risk Gene Signature Assay for Predicting Breast Cancer Risk.

BACKGROUND: Breast cancer (BC) risk assessment models are statistical estimates based on patient characteristics. We developed a gene expression assay to assess BC risk using benign breast biopsy tissue. METHODS: A NanoString-based malignancy risk (MR) gene signature was validated for formalin-fixed paraffin-embedded (FFPE) tissue. It was applied to FFPE benign and BC specimens obtained from women who underwent breast biopsy, some of whom developed BC during follow-up to evaluate diagnostic capability of the MR signature. BC risk was calculated with MR score, Gail risk score, and both tests combined. Logistic regression and receiver operating characteristic curves were used to evaluate these 3 models. RESULTS: NanoString MR demonstrated concordance between fresh frozen and FFPE malignant samples (r = 0.99). Within the validation set, 563 women with benign breast biopsies from 2007 to 2011 were identified and followed for at least 5 y; 50 women developed BC (affected) within 5 y from biopsy. Three groups were compared: benign tissue from unaffected and affected patients and malignant tissue from affected patients. Kruskal-Wallis test suggested difference between the groups (P = 0.09) with trend in higher predicted MR score for benign tissue from affected patients before development of BC. Neither the MR signature nor Gail risk score were statistically different between affected and unaffected patients; combining both tests demonstrated best predictive value (AUC = 0.71). CONCLUSIONS: FFPE gene expression assays can be used to develop a predictive test for BC. Further investigation of the combined MR signature and Gail Model is required. Our assay was limited by scant cellularity of archived breast tissue.

SinCHet: a MATLAB toolbox for single cell heterogeneity analysis in cancer.

SUMMARY: Single-cell technologies allow characterization of transcriptomes and epigenomes for individual cells under different conditions and provide unprecedented resolution for researchers to investigate cellular heterogeneity in cancer. The SinCHet ( gle ell erogeneity) toolbox is developed in MATLAB and has a graphical user interface (GUI) for visualization and user interaction. It analyzes both continuous (e.g. mRNA expression) and binary omics data (e.g. discretized methylation data). The toolbox does not only quantify cellular heterogeneity using S hannon P rofile (SP) at different clonal resolutions but also detects heterogeneity differences using a D statistic between two populations. It is defined as the area under the P rofile of S hannon D ifference (PSD). This flexible tool provides a default clonal resolution using the change point of PSD detected by multivariate adaptive regression splines model; it also allows user-defined clonal resolutions for further investigation. This tool provides insights into emerging or disappearing clones between conditions, and enables the prioritization of biomarkers for follow-up experiments based on heterogeneity or marker differences between and/or within cell populations. AVAILABILITY AND IMPLEMENTATION: The SinCHet software is freely available for non-profit academic use. The source code, example datasets, and the compiled package are available at http://labpages2.moffitt.org/chen/software/ . CONTACT: ann.chen@moffitt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

APOSTL: An Interactive Galaxy Pipeline for Reproducible Analysis of Affinity Proteomics Data.

With continuously increasing scale and depth of coverage in affinity proteomics (AP-MS) data, the analysis and visualization is becoming more challenging. A number of tools have been developed to identify high-confidence interactions; however, a cohesive and intuitive pipeline for analysis and visualization is still needed. Here we present Automated Processing of SAINT Templated Layouts (APOSTL), a freely available Galaxy-integrated software suite and analysis pipeline for reproducible, interactive analysis of AP-MS data. APOSTL contains a number of tools woven together using Galaxy workflows, which are intuitive for the user to move from raw data to publication-quality figures within a single interface. APOSTL is an evolving software project with the potential to customize individual analyses with additional Galaxy tools and widgets using the R web application framework, Shiny. The source code, data, and documentation are freely available from GitHub ( https://github.com/bornea/APOSTL ) and other sources.

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms.

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.

Phase Ib trial of IRX-2 plus durvalumab in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

OBJECTIVES: IRX-2 is a multi-cytokine immune-activating agent with anti-tumor activity in non-metastatic head and neck squamous cell carcinoma (HNSCC). Here, we evaluated combined IRX-2 and durvalumab in patients with recurrent and/or metastatic HNSCC. MATERIALS AND METHODS: This was a phase Ib trial consisting of dose escalation and expansion. Primary endpoints were safety and biomarkers to assess the immune response in the tumor microenvironment including significant increases in PD-L1 expression and CD8 + tumor infiltrating lymphocytes (TIL) comparing pre- and on-treatment tumor biopsies. Secondary endpoints were objective response rates (ORR) and survival outcomes. RESULTS: Sixteen patients were evaluable for response, and nine patients were evaluable for biomarkers. Thirteen patients (68 %) had exposure to prior anti-PD-1 therapy. No dose-limiting or grade ≥ 3 treatment-related adverse events were observed. On-treatment biopsies showed significantly increased PD-L1 (p = 0.005), CD3+ (p = 0.020), CD4+ (p = 0.022), and CD8 + T cells (p = 0.017) compared to pre-treatment. Median overall survival and progression-free survival (PFS) were 6.18 months (95 % CI, 2.66-8.61) and 2.53 months (95 % CI, 1.81-4.04), respectively. One patient had an objective response (ORR, 5.3 %) with an ongoing PFS of > 25 months. Disease control rate was 42 %. The responder harbored an ARID1A variant of unknown significance (VUS) that was predicted to bind her HLA-I alleles with a higher affinity than the reference peptide. CONCLUSIONS: IRX-2 and durvalumab were safe and elicited the evidence of immune activation in the tumor microenvironment determined by increased PD-L1 expression and CD8+ TILs. CLINICAL TRIAL REGISTRATION NUMBER: NCT03381183.

Pancreatic Cyst Size Measurement on Magnetic Resonance Imaging Compared to Pathology.

BACKGROUND: While multiple cyst features are evaluated for stratifying pancreatic intraductal papillary mucinous neoplasms (IPMN), cyst size is an important factor that can influence treatment strategies. When magnetic resonance imaging (MRI) is used to evaluate IPMNs, no universally accepted sequence provides optimal size measurements. T2-weighted coronal/axial have been suggested as primary measurement sequences; however, it remains unknown how well these and maximum all-sequence diameter measurements correlate with pathology size. This study aims to compare agreement and bias between IPMN long-axis measurements on seven commonly obtained MRI sequences with pathologic size measurements. METHODS: This retrospective cohort included surgically resected IPMN cases with preoperative MRI exams. Long-axis diameter tumor measurements and the presence of worrisome features and/orhigh-risk stigmata were noted on all seven MRI sequences. MRI size and pathology agreement and MRI inter-observer agreement involved concordance correlation coefficient (CCC) and intraclass correlation coefficient (ICC), respectively. The presence of worrisome features and high-risk stigmata were compared to the tumor grade using kappa analysis. The Bland-Altman analysis assessed the systematic bias between MRI-size and pathology. RESULTS: In 52 patients (age 68 ± 13 years, 22 males), MRI sequences produced mean long-axis tumor measurements from 2.45-2.65 cm. The maximum MRI lesion size had a strong agreement with pathology (CCC = 0.82 (95% CI: 0.71-0.89)). The maximum IPMN size was typically observed on the axial T1 arterial post-contrast and MRCP coronal series and overestimated size versus pathology with bias +0.34 cm. The radiologist interobserver agreement reached ICCs 0.74 to 0.91 on the MRI sequences. CONCLUSION: The maximum MRI IPMN size strongly correlated with but tended to overestimate the length compared to the pathology, potentially related to formalin tissue shrinkage during tissue processing.

A Real-World Data Retrospective Cohort Study of HER2-positive, Early-Stage Breast Cancer in Patients 70 Years of Age or Older: Natural History, Treatment Patterns, and Outcomes.

PURPOSE: Early-stage human epidermal growth factor receptor 2-positive (HER2-positive) breast cancers (BCs) are routinely treated with intense perioperative chemotherapy combined with HER2-targeted agents. There is thus an unmet need for knowledge about treatment patterns and outcomes among patients 70 years of age or older, as this is an under-represented subset of patients in large clinical trials. METHODS: We used a deidentified cohort derived from a nationwide electronic health record database to conduct a retrospective cohort study of patients with HER2-positive BCs. Descriptive statistics were used to evaluate tumor characteristics, treatment patterns across age groups, and pathologic complete response rates. We used Kaplan-Meier survival curves to estimate recurrence-free and overall survivals; Cox proportional methods were used for adjustments with covariates of interest, including age as a categorical variable. RESULTS: We included 395 patients with HER2-positive stage I to III BCs who were 70 years of age or older. Most patients had tumors with high nuclear-grade T2 tumors, and received surgical treatment first. Most patients (61.7%) who received HER2 therapies underwent treatment in the adjuvant setting; paclitaxel and trastuzumab combination was the most commonly used adjuvant regimen. Older age was associated with increased hazard of recurrence or death. We did not detect significant evidence of decline in performance status, but there was modest weight drop after perioperative HER2 treatments. CONCLUSION: Findings suggest that patients in this older-age cohort were treated with de-escalated perioperative strategies and had poorer outcomes; our findings should be validated in future studies.

The Polyamine-Hypusine Circuit Controls an Oncogenic Translational Program Essential for Malignant Conversion in MYC-Driven Lymphoma.

The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given this, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine-hypusine circuit, which posttranslationally modifies the eukaryotic translation factor eIF5A. The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where the loss of eIF5A hypusination abolishes malignant transformation of MYC-overexpressing B cells. Mechanistically, integrating RNA sequencing, ribosome sequencing, and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1-S phase cell-cycle progression and DNA replication. This circuit thus controls MYC's proliferative response, and it is also activated across multiple malignancies. These findings suggest the hypusine circuit as a therapeutic target for several human tumor types. SIGNIFICANCE: Elevated EIF5A and the polyamine-hypusine circuit are manifest in many malignancies, including MYC-driven tumors, and eIF5A hypusination is necessary for MYC proliferative signaling. Not-ably, this circuit controls an oncogenic translational program essential for the development and maintenance of MYC-driven lymphoma, supporting this axis as a target for cancer prevention and treatment. See related commentary by Wilson and Klein, p. 248. This article is highlighted in the In This Issue feature, p. 247.

Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

BACKGROUND: Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. METHODS: We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination. RESULTS: The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. CONCLUSIONS: Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

Dose-Limiting Pulmonary Toxicity in a Phase 1/2 Study of Radiation and Chemotherapy with Ipilimumab Followed by Nivolumab for Patients With Stage 3 Unresectable Non-Small Cell Lung Cancer.

PURPOSE: We hypothesized that concurrent ipilimumab with chemoradiationtherapy (chemoRT) followed by maintenance nivolumab would be safe for patients with unresectable stage III non-small cell lung cancer (NSCLC). We aimed to assess the safety (phase 1) and the 12-month progression-free survival (PFS) (phase 2) in a multi-institution prospective trial. METHODS AND MATERIALS: Eligible patients had unresectable stage III NSCLC. The treatment included platinum doublet chemotherapy with concurrent thoracic radiation therapy to 60 Gy in 30 fractions and ipilimumab (1 mg/kg) delivered during weeks 1 and 4. After chemoRT, maintenance nivolumab (480 mg) was given every 4 weeks for up to 12 cycles. Adverse events (AEs) were assessed according to the Common Terminology Criteria for Adverse Events, version 5.0. Survival analyses were performed with Kaplan Meier (KM) methods and log-rank tests. RESULTS: The trial was discontinued early after enrolling 19 patients without proceeding to the phase 2 component because of unacceptable toxicity. Sixteen patients (84%) had grade ≥3 (G3+) possible treatment-related toxicity, most commonly pulmonary AEs (n = 8, 42%). Fourteen patients (74%) discontinued study therapy early because of AEs (n = 12, 63%) or patient choice (n = 2, 11%). Eleven patients (58%) experienced G2+ pulmonary toxicity with median time to onset 4.1 months (95% CI 2.6-not reached [NR]), and 12-month freedom from G2+ pulmonary toxicity 37% (95% CI, 16-59). Five patients had G5 AEs, including 3 with G5 pulmonary AEs (1 respiratory failure with pneumonitis and pulmonary embolism, 1 pneumonia/chronic obstructive pulmonary disease exacerbation, 1 pulmonary fibrosis). Despite toxicities, the median PFS was 19.2 months (95% CI 6.1-NR) and the median overall survival was NR (95% CI 6.1-NR) with median follow-up of 30.1 months by the reverse KM method. CONCLUSIONS: Concurrent ipilimumab with chemoRT for unresectable stage III NSCLC is associated with pulmonary toxicity that may limit opportunities for improved outcomes. Future studies aiming to incorporate ipilimumab or other anti-CTLA4 therapies into management of unresectable stage III NSCLC should consider careful measures to minimize toxicity risk.

A chemical and phosphoproteomic characterization of dasatinib action in lung cancer.

We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.

A Real-World Data Retrospective Cohort Study of Low Estrogen Receptor-Positive Early Breast Cancer: Natural History and Treatment Outcomes.

Purpose: Estrogen receptor-positive (ER+) breast cancer (BC) is a heterogeneous disease, and there is an ongoing debate regarding the optimal cut point for clinically relevant ER expression. We used a real-world database to assess the prognostic and predictive values of lower ER expression levels on treatment outcomes with endocrine therapy. Methods: We used a nationwide electronic health record database. Descriptive statistics were used to evaluate the association between ER expression, tumor characteristics, and treatment patterns among patients with early-stage BC. We used Kaplan-Meier survival curves to estimate recurrence-free survival (RFS) and overall survival (OS). We assessed associations between an alternative ER expression-level cut point and clinical outcomes. Results: Among 4697 patients with early-stage HER2-negative BC, 83 (2.04%) had ER+-low BC (ER expression, 1-9.99%) and 36 (0.88%) had ER+-intermediate BC (10-19.9%). ER+-low tumors were associated with higher tumor grade, larger size, and higher axillary tumor burden than ER+-high tumors (≥20% ER expression). African Americans had a higher prevalence of both triple-negative BC (TNBC) and ER+-low BC than ER+-high BC. Patients with ER+-low and ER+-intermediate tumors had survival outcomes similar to patients with TNBC and worse survival outcomes than patients with ER+-high tumors (P < 0.001). Tumors with <20% ER expression were associated with worse outcomes. Conclusion: In our cohort, patients with BCs with ER expression levels <20% had poor clinical outcomes similar to those of patients with TNBC.

Adding Cyclooxygenase Inhibitors to Immune Checkpoint Inhibitors Did Not Improve Outcomes in Metastatic Renal Cell Carcinoma.

Modulating the cyclooxygenase 2 (COX-2) pathway has improved responses to immune checkpoint inhibitors (ICIs) in certain solid tumors, such as melanoma. Little is known about COX-2 inhibition in response to ICIs in metastatic renal cell carcinoma (mRCC). In this retrospective cohort study, we examined the effect of COX-2 inhibitors on the long-term outcomes of mRCC patients undergoing ICI therapies. Among 211 patients with mRCC, 23 patients were excluded due to loss to follow-up. Among 188 included patients, 120 patients received either an NSAID or aspirin for at least three weeks during ICI therapies. Clear cell histology was present in 96% of cases. The median overall survival (OS) was similar regardless of the COX inhibitor (COXi) (i.e., NSAID or aspirin) use (27 months for COXi vs. 33 months for no-COXi groups; p = 0.73). The no-COXi group showed a trend toward longer median progression-free survival (8 months for COXi vs. 13 months for no-COXi groups; p = 0.13). When looking specifically at NSAID use in a multivariate analysis, NSAID use was associated with a higher risk of progression (HR = 1.52 [95% CI, 1.04-2.22]) and death (HR = 1.60 [95% CI, 1.02-2.52]). In summary, COXis did not improve disease control or survival among patients with mRCC who were undergoing ICI therapies. Instead, the concurrent use of NSAIDs was associated with worse outcomes. Larger studies are needed to validate our observation.

Drepmel-A Multi-Omics Melanoma Drug Repurposing Resource for Prioritizing Drug Combinations and Understanding Tumor Microenvironment.

Although substantial progress has been made in treating patients with advanced melanoma with targeted and immuno-therapies, de novo and acquired resistance is commonplace. After treatment failure, therapeutic options are very limited and novel strategies are urgently needed. Combination therapies are often more effective than single agents and are now widely used in clinical practice. Thus, there is a strong need for a comprehensive computational resource to define rational combination therapies. We developed a Shiny app, DRepMel to provide rational combination treatment predictions for melanoma patients from seventy-three thousand combinations based on a multi-omics drug repurposing computational approach using whole exome sequencing and RNA-seq data in bulk samples from two independent patient cohorts. DRepMel provides robust predictions as a resource and also identifies potential treatment effects on the tumor microenvironment (TME) using single-cell RNA-seq data from melanoma patients. Availability: DRepMel is accessible online.