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RAF inhibitor “paradoxical activation” (PA) is a phenomenon where RAF kinase inhibitors increase RAF kinase signaling. Through mathematical modeling and experimental data analysis, we recently demonstrated that the combination of conformational autoinhibition (CA) with the disruption of CA by RAF inhibitors plays an important role in PA. 14-3-3 proteins are known to modulate RAF CA and RAF dimerization. We here extend our mathematical model to include both roles of 14-3-3 proteins, and we derive rigorous analytical expressions of RAF signal regulation as modulated by 14-3-3 proteins. We then use the model to investigate how 14-3-3 proteins may modulate PA. We mathematically show 14-3-3 protein stabilization of the autoinhibited form of RAF should potentiate PA, while 14-3-3 protein stabilization of the active RAF dimer should reduce PA. Our analysis suggests that the net-effect will often be a potentiation of PA, and that 14-3-3 proteins may be capable of inducing PA for RAF inhibitors that normally show little to no PA. We test model-based insights experimentally with two different approaches: forced increases in 14-3-3 expression (which we find amplifies PA) and evolved resistance assays (which suggest increased 14-3-3 expression may contribute to resistance to RAF inhibitors). Overall, this work supports a role for 14-3-3 in modulating RAF-inhibitor mediated paradoxical activation.

Phase three clinical trial evidence suggests that colorectal cancers with the KRAS G13D mutation may benefit from EGFR inhibitors, like cetuximab, in contrast to the other most common KRAS mutations. A mechanism to explain why this mutation behaves differently from other KRAS mutations had long been lacking. Two recent studies have reproduced KRAS G13D specific sensitivity to cetuximab in cellular models, and both have implicated the tumor suppressor NF1 as a critical variable in determining sensitivity and resistance. One study proposes a mechanism that focuses on the inhibition of active, GTP-bound wild-type RAS, which is proposed to occur to a greater extent in KRAS G13D tumors due to the inability of KRAS G13D to bind NF1 well. The other study suggests NF1 can convert GTP-bound KRAS G13D to inactive, GDP-bound KRAS G13D. Here, we report an inability to reproduce cellular and biophysical studies that suggested NF1 has strong GTPase activity on KRAS G13D. We also report additional data that further suggests only WT RAS-GTP levels are reduced with EGFR inhibition and that KRAS G13D is impaired in binding to NF1. These new experiments further support a mechanism in which cetuximab inhibits wild-type (HRAS and NRAS) signals in KRAS G13D colorectal cancers. Dm4iGs5nWD8FkGrYCfJL2M Video Abstract

The combination of KRAS G12C inhibitors with EGFR inhibitors has reproducibly been shown to be beneficial. Here, we identify another benefit of this combination: it effectively inhibits both wild-type and mutant RAS. We believe that targeting both mutant and wild-type RAS helps explain why this combination of inhibitors is effective.

Pancreatic Ductal Adenocarcinoma (PDAC) is often caused by mutations in multiple genes including KRAS (activating the Ras-Raf-MEK-ERK pathway). This study evaluated the role of MEK inhibitor (MEKi)-based combinatorial targeted therapies in patients with PDAC. Methods. This is a retrospective/prospective observational, single institution study, including 29 patients with metastatic PDAC with KRAS alterations, treated with MEKi therapies between 2022-2024. Ten patients had KRAS G12R (34.5%), ten G12D (34.5%), and nine G12V (31%). Majority of patients received MEKi therapy as third-line and beyond (KRAS G12R/G12D/G12V 60%/50%/78%, respectively). Median overall survival from MEKi initiation for KRAS G12R/G12D/G12V was 8.2/5.1/4.7 months (P = 0.5), respectively, and median progression-free survival was 4.4/2.3/1.4 months (P = 0.11). Six (21%) patients discontinued at least one drug in the treatment combination due to toxicity. MEKi-based combinatorial therapies had modest disease control in patients with KRAS G12R, and minimal disease control in patients with KRAS G12D/V in the late-line setting.

Background: KRAS wild-type (WT) pancreatic ductal adenocarcinoma (PDAC) represents a distinct entity with unique biology. The therapeutic impact of matched targeted therapy in these patients in a real-world setting, to date, is less established. Objectives: The aim of our study was to review our institutional database to identify the prevalence of actionable genomic alterations in patients with KRAS-WT tumors and to evaluate the therapeutic impact of matched targeted therapy in these patients. Design: We reviewed electronic medical records of patients with KRAS-WT PDAC and advanced disease (n = 14) who underwent clinical-grade tissue ± liquid next-generation sequencing (315–648 genes for tissue) between years 2015 and 2021. Methods: Demographic and disease characteristics were summarized using descriptive parameters. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method. Results: Of 236 PDAC patients, 14 had advanced/metastatic disease with KRAS-WT tumors. Median age at diagnosis was 66 years. There was a high frequency of potentially actionable genomic alterations, including three (21%) with BRAF alterations, two (14%) with fusions [RET-PCM1 and FGFR2-POC1B (N = 1 each)]; and one with a druggable EGFR (EGFR E746_A755delISERD) variant; two other patients had an STK11 and a MUTYH alteration. Five patients were treated with matched targeted therapy, with three having durable benefit: (i) erlotinib for EGFR-altered tumor, followed by osimertinib/capmatinib when MET amplification emerged (first-line therapy); (ii) pralsetinib for RET fusion (fifth line); and (iii) dabrafenib/trametinib for BRAF N486_P490del (third line). Duration of time on chemotherapy-free matched targeted therapy for these patients was 17+, 11, and 18+ months, respectively. Conclusion: Sustained therapeutic benefit can be achieved in a real-world setting in a subset of patients with advanced/metastatic KRAS-WT PDAC treated with chemotherapy-free matched targeted agents. Prospective studies are warranted.

Dexamethasone (Dex), co-administered to lung adenocarcinoma patients with pemetrexed chemotherapy, protects against pemetrexed cytotoxicity by inducing reversible G1 arrest, reflected by the effect of Dex on FLT-PET images of patient tumors. However, perioperative Dex treatment increases survival but the mechanism is unknown. In cells with glucocorticoid receptor-α (GR) expression corresponding to higher clinical tumor levels, Dex-induced growth arrest was followed by marked cell expansion, beta-galactosidase expression and Ki67 negativity, despite variable p53 and K-RAS status. Dex induced a transient early surge in p21 Cip1 . However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21 Cip1 and caused by gradual accumulation of p27 Kip1 due to transcriptional activation of p27 Kip1 by Dex. This effect was independent of canonical pathways of senescence or p27 Kip1 regulation. The in vitro observations were reflected by growth suppression and P27 Kip1 induction in GR-overexpressing tumor xenografts compared with isogenic low-GR tumors. Extended Dex treatment induces irreversible cell cycle blockade and a senescence phenotype through chronic activation of the p27 Kip1 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.

Background: KRAS wild-type (WT) pancreatic ductal adenocarcinoma (PDAC) represents a distinct entity with unique biology. The therapeutic impact of matched targeted therapy in these patients in a real-world setting, to date, is less established. Objectives: The aim of our study was to review our institutional database to identify the prevalence of actionable genomic alterations in patients with KRAS -WT tumors and to evaluate the therapeutic impact of matched targeted therapy in these patients. Design: We reviewed electronic medical records of patients with KRAS-WT PDAC and advanced disease ( n = 14) who underwent clinical-grade tissue ± liquid next-generation sequencing (315–648 genes for tissue) between years 2015 and 2021. Methods: Demographic and disease characteristics were summarized using descriptive parameters. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method. Results: Of 236 PDAC patients, 14 had advanced/metastatic disease with KRAS-WT tumors. Median age at diagnosis was 66 years. There was a high frequency of potentially actionable genomic alterations, including three (21%) with BRAF alterations, two (14%) with fusions [ RET-PCM1 and FGFR2-POC1B ( N = 1 each)]; and one with a druggable EGFR ( EGFR E746_(A)755delISERD) variant; two other patients had an STK11 and a MUTYH alteration. Five patients were treated with matched targeted therapy, with three having durable benefit: (i) erlotinib for EGFR -altered tumor, followed by osimertinib/capmatinib when MET amplification emerged (first-line therapy); (ii) pralsetinib for RET fusion (fifth line); and (iii) dabrafenib/trametinib for BRAF N486_(P)490del (third line). Duration of time on chemotherapy-free matched targeted therapy for these patients was 17+, 11, and 18+ months, respectively. Conclusion: Sustained therapeutic benefit can be achieved in a real-world setting in a subset of patients with advanced/metastatic KRAS-WT PDAC treated with chemotherapy-free matched targeted agents. Prospective studies are warranted.

Over 20% of breast cancer cases present with distal metastasis and they are predominantly of luminal subtypes. As luminal breast cancer is relatively indolent, it is believed that progression to metastasis must occur over many years, generally well into post-menopausal years. Unfortunately, very little is known about the mechanisms by which these hormone receptor positive tumors metastasize, likely in part due to their slow metastatic rates in animal model systems as well. Moreover, the literature lacks adequate mechanistic understanding of cross talk between estradiol (E2) and progesterone, particularly in the context of breast cancer invasion and metastasis. In this thesis, we sought to investigate the roles of estrogen and progesterone and their nuclear receptors to better understand hormonal regulation of metastasis at physiologically relevant hormone levels both pre- and post-menopause. The novelty of our experimental approach and study design is three-fold: 1. exploration of the isoform-specific actions of the progesterone receptor; 2. investigation of selective micro RNA mediated pathways of cross talk between estrogen and progesterone and 3. development of a quantitative lymph node infiltration assay to monitor metastasis of luminal breast cancer in xenograft models.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, comprising 80% of all malignant gliomas. Recently, active Human Cytomegalovirus (HCMV) was identified in GBM cells, and has been a topic of debate concerning its role with tumor progression. This study used three established GBM cell lines; T98, LN229, and U87 in order to identify and examine the presence of HCMV phosphotransferase protein UL97. Reverse transcriptase polymerase chain reaction identified UL97 within two of the three cell lines, T98 and LN229. Western blotting confirmed that UL97 protein was being expressed and was present in both T98, and LN229 cell lines. UL97 is a phosphotransferase that has the ability to phosphorylate guanosine analogues, creating a guanosine triphosphate that inhibits DNA elongation and replication. Ganciclovir, a guanosine analogue, was used to treat GBM cell lines and our results demonstrate that it significantly decreases cellular proliferation in UL97 expressing cells. This project identifies a new role for HCMV in GBM and suggests a possible future treatment option.

Dexamethasone (Dex), co-administered to lung adenocarcinoma patients with pemetrexed chemotherapy, protects against pemetrexed cytotoxicity by inducing reversible G1 arrest, reflected by the effect of Dex on FLT-PET images of patient tumors. However, perioperative Dex treatment increases survival but the mechanism is unknown. In cells with glucocorticoid receptor-α (GR) expression corresponding to higher clinical tumor levels, Dex-induced growth arrest was followed by marked cell expansion, beta-galactosidase expression and Ki67 negativity, despite variable p53 and K-RAS status. Dex induced a transient early surge in p21 . However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21 and caused by gradual accumulation of p27 due to transcriptional activation of p27 by Dex. This effect was independent of canonical pathways of senescence or p27 regulation. The in vitro observations were reflected by growth suppression and P27 induction in GR-overexpressing tumor xenografts compared with isogenic low-GR tumors. Extended Dex treatment induces irreversible cell cycle blockade and a senescence phenotype through chronic activation of the p27 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.