Transcriptomic-Based Microenvironment Classification Reveals Precision Medicine Strategies for Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The complex tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) has hindered the development of reliable predictive biomarkers for targeted therapy and immunomodulatory strategies. A comprehensive characterization of the TME is necessary to advance precision therapeutics in PDAC. METHODS: A transcriptomic profiling platform for TME classification based on functional gene signatures was applied to 14 publicly available PDAC datasets (n = 1657) and validated in a clinically annotated independent cohort of patients with PDAC (n = 79). Four distinct subtypes were identified using unsupervised clustering and assessed to evaluate predictive and prognostic utility. RESULTS: TME classification using transcriptomic profiling identified 4 biologically distinct subtypes based on their TME immune composition: immune enriched (IE); immune enriched, fibrotic (IE/F); fibrotic (F); and immune depleted (D). The IE and IE/F subtypes demonstrated a more favorable prognosis and potential for response to immunotherapy compared with the F and D subtypes. Most lung metastases and liver metastases were subtypes IE and D, respectively, indicating the role of clonal phenotype and immune milieu in developing personalized therapeutic strategies. In addition, distinct TMEs with potential therapeutic implications were identified in treatment-naive primary tumors compared with tumors that underwent neoadjuvant therapy. CONCLUSIONS: This novel approach defines a distinct subgroup of PADC patients that may benefit from immunotherapeutic strategies based on their TME subtype and provides a framework to select patients for prospective clinical trials investigating precision immunotherapy in PDAC. Further, the predictive utility and real-world clinical applicability espoused by this transcriptomic-based TME classification approach will accelerate the advancement of precision medicine in PDAC.

KRAS-Mutated, Estrogen Receptor-Positive Low-Grade Serous Ovarian Cancer: Unraveling an Exceptional Response Mystery.

We report on a woman with aggressive estrogen receptor-positive, KRAS-mutated ovarian cancer who achieved a remarkable response to combination therapy with the MEK inhibitor (trametinib) and the aromatase inhibitor (letrozole), even though the disease had failed to respond to a combination of a PI3K inhibitor and different MEK inhibitor, as well as to trametinib and the estrogen modulator, tamoxifen, and to letrozole by itself. The mechanism of action for exceptional response was elucidated by in vitro experiments that demonstrated that the fact that tamoxifen can have an agonistic effect in addition to antagonist activity, whereas letrozole results only in estrogen depletion was crucial to the response achieved when letrozole was combined with an MEK inhibitor. Our current observations indicate that subtle variations in mechanisms of action of outwardly similar regimens may have a major impact on outcome and that such translational knowledge is critical for optimizing a precision medicine strategy. KEY POINTS: This report describes the remarkable response of a patient with KRAS-mutated, estrogen receptor-positive low-grade serous ovarian cancer treated with trametinib (MEK inhibitor) and letrozole (aromatase inhibitor), despite prior progression on similar agents including tamoxifen (estrogen modulator). In vitro investigation revealed that tamoxifen can have agonistic in addition to antagonistic effects, which could be the reason for the patient not responding to the combination of trametinib and tamoxifen. The current observations suggest that drugs with different mechanisms of action targeting the same receptor may have markedly different anticancer activity when used in combinations.

Discernment between candidate mechanisms for KRAS G13D colorectal cancer sensitivity to EGFR inhibitors.

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. Video Abstract.

Progesterone receptor A promotes invasiveness and metastasis of luminal breast cancer by suppressing regulation of critical microRNAs by estrogen.

Distal metastasis of luminal breast cancer is frequent and incurable, yet the metastasis mechanisms are poorly understood. Estrogen, even at postmenopausal concentrations, suppresses invasiveness of luminal breast cancer cells through the estrogen receptor (ER). Invasive tumors overexpress the short progesterone receptor A (PR-A) isoform. Even at postmenopausal concentrations, progesterone activates PR-A, inducing invasiveness by counteracting estrogen's effects, particularly when cells are hypersensitized to progesterone by PR-A overexpression. To interrogate the role of this cross-talk in metastasis, we investigated selective cross-talk mechanisms of PR-A with ER. We developed a quantitative PCR-based lymph node infiltration assay to address the slowness of metastasis of tumor xenografts. We found that 15 microRNAs (miRNAs) are regulated by progesterone via PR-A, but not the longer PR-B isoform, with increased progesterone sensitivity when PR-A was overexpressed. Two of these miRNAs whose induction (miR-92a-3p) or repression (miR-26b-5p) by estrogen was suppressed by progesterone plus PR-A were critical for the PR-A-ER cross-talk causing a gene-regulatory pattern of invasiveness and metastasis and complete rescue of invasiveness in vitro Constitutive expression of miR-92a-3p or inhibition of miR-26b-5p profoundly suppressed metastasis. Finally, in primary breast tumors, PR-A expression was correlated negatively with miR-92a-3p expression and positively with miR-26b-5p expression. Therefore, hormonal cross-talk of PR-A with ER is probably a fundamental mechanism that enables metastasis of luminal breast cancer. Moreover, miRNA biomarkers of hyperactive PR-A may help predict metastatic potential of luminal breast tumors. Further, miR-92a-3p and miR-26b-5p may reveal target pathways for selective intervention to suppress hormone-regulated metastasis, both pre- and postmenopause.

The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth.

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10-8 m A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells.

Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCa) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCa treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamide-resistant PCa cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate α-tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulate HSP90 and AR in enzalutamide-resistant PCa cells with weakened effects on nuclear HDACI targets.

Chemotherapy-free treatment targeting fusions and driver mutations in KRAS wild-type pancreatic ductal adenocarcinoma, a case series.

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.

Co-targeting KRAS G12C and EGFR reduces both mutant and wild-type RAS-GTP.

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.

Identification of RAS mutant biomarkers for EGFR inhibitor sensitivity using a systems biochemical approach.

Mutations can be important biomarkers that influence the selection of specific cancer treatments. We recently combined mathematical modeling of RAS signaling network biochemistry with experimental cancer cell biology to determine why KRAS G13D is a biomarker for sensitivity to epidermal growth factor receptor (EGFR)-targeted therapies. The critical mechanistic difference between KRAS G13D and the other most common KRAS mutants is impaired binding to tumor suppressor Neurofibromin (NF1). Here, we hypothesize that impaired binding to NF1 is a "biophysical biomarker" that defines other RAS mutations that retain therapeutic sensitivity to EGFR inhibition. Both computational and experimental investigations support our hypothesis. By screening RAS mutations for this biophysical characteristic, we identify 10 additional RAS mutations that appear to be biomarkers for sensitivity to EGFR inhibition. Altogether, this work suggests that personalized medicine may benefit from migrating from gene-based and allele-based biomarker strategies to biomarkers based on biophysically defined subsets of mutations.

A mechanism for the response of KRASG13D expressing colorectal cancers to EGFR inhibitors.

Previous analysis of Phase 3 clinical trial data for colorectal cancer patients treated with cetuximab revealed that patients harboring a KRAS mutation did not benefit from treatment. This finding set the stage for one of the first examples of cancer personalized medicine. Confusingly, patients with a Glycine to Aspartic Acid mutation at amino acid 13 of KRAS (KRASG13D) appeared to respond positively to cetuximab, suggesting this mutation is an exception to the rule that KRAS mutations confer resistance to Epidermal Growth Factor Receptor (EGFR) inhibitors. Oncologists have stated that the mechanism that explains why the KRASG13D mutation is an exception should be identified before KRASG13D colorectal cancer patients should be treated differently. We have recently elucidated this mechanism using mathematical modeling of the KRAS biochemical system coupled with experimental biology. The mechanism we revealed involves a cetuximab-mediated reduction in HRAS and NRAS signaling within KRASG13D cancer cells, owing to impaired binding of KRASG13D to the tumor suppressor, Neurofibromin (NF1).

A systems mechanism for KRAS mutant allele-specific responses to targeted therapy.

Cancer treatment decisions are increasingly guided by which specific genes are mutated within each patient's tumor. For example, agents inhibiting the epidermal growth factor receptor (EGFR) benefit many colorectal cancer (CRC) patients, with the general exception of those whose tumor includes a KRAS mutation. However, among the various KRAS mutations, that which encodes the G13D mutant protein (KRASG13D) behaves differently; for unknown reasons, KRASG13D CRC patients benefit from the EGFR-blocking antibody cetuximab. Controversy surrounds this observation, because it contradicts the well-established mechanisms of EGFR signaling with regard to RAS mutations. Here, we identified a systems-level, mechanistic explanation for why KRASG13D cancers respond to EGFR inhibition. A computational model of RAS signaling revealed that the biophysical differences between the three most common KRAS mutants were sufficient to generate different sensitivities to EGFR inhibition. Integrated computation with experimentation then revealed a nonintuitive, mutant-specific dependency of wild-type RAS activation by EGFR that is determined by the interaction strength between KRAS and the tumor suppressor neurofibromin (NF1). KRAS mutants that strongly interacted with and competitively inhibited NF1 drove wild-type RAS activation in an EGFR-independent manner, whereas KRASG13D weakly interacted with and could not competitively inhibit NF1 and, thus, KRASG13D cells remained dependent on EGFR for wild-type RAS activity. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine and can help identify patients for selective therapeutic strategies.

Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor.

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 p21Cip1. However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21Cip1and caused by gradual accumulation of p27Kip1 due to transcriptional activation of p27Kip1 by Dex. This effect was independent of canonical pathways of senescence or p27Kip1 regulation. The in vitro observations were reflected by growth suppression and P27Kip1 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 p27Kip1 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

PURPOSE: Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. EXPERIMENTAL DESIGN: Small-molecule drug discovery and extensive biological characterization of a lead compound. RESULTS: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. CONCLUSIONS: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.

Role of the short isoform of the progesterone receptor in breast cancer cell invasiveness at estrogen and progesterone levels in the pre- and post-menopausal ranges.

Overexpression of the progesterone receptor (PR) isoform A (PR-A) is a negative prognosticator for estrogen receptor (ER)-positive breast cancer but in vitro studies have implicated PR-B in progestin-induced invasiveness. As estrogen is known to suppress invasiveness and tumor progression and as the in vitro studies were conducted in models that either lacked ER or excluded estrogen, we examined the role of PR isoforms in the context of estrogen signaling. Estrogen (< 0.01nM) strongly suppressed invasiveness in various ER+ model cell lines. At low (< 1nM) concentrations, progestins completely abrogated inhibition of invasiveness by estrogen. It was only in a higher (5 nM - 50 nM) concentration range that progestins induced invasiveness in the absence of estrogen. The ability of low dose progestins to rescue invasiveness from estrogen regulation was exclusively mediated by PR-A, whereas PR-B mediated the estrogen-independent component of progestin-induced invasiveness. Overexpression of PR-A lowered the progestin concentration needed to completely rescue invasiveness. Among estrogen-regulated genes, progestin/PR-A counter-regulated a distinctive subset, including breast tumor progression genes (e.g., HES1, PRKCH, ELF5, TM4SF1), leading to invasiveness. In this manner, at relatively low hormone concentrations (corresponding to follicular stage and post-menopausal breast tissue or plasma levels), progesterone influences breast cancer cell invasiveness by rescuing it from estrogen regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of estrogen signaling, through PR-B. The findings point to a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue hormone levels.

Glucocorticoid receptor status is a principal determinant of variability in the sensitivity of non-small-cell lung cancer cells to pemetrexed.

INTRODUCTION: Pemetrexed is an S-phase targeted drug in front-line or maintenance therapy of advanced nonsquamous non-small-cell lung cancer (NSCLC) but methods are needed for predicting the drug response. Dexamethasone is typically administered the day before, the day of, and the day after pemetrexed. As dexamethasone strongly regulates many genes including p53 through the glucocorticoid receptor (GR), we hypothesized that dexamethasone influences tumor response to pemetrexed. METHODS: Eight nonsquamous NSCLC cell line models with varied p53 and GRα/GRß status were used for gene expression and cell-cycle analyses and for loss- or gain-of-function experiments. RESULTS: In three cell lines dexamethasone profoundly, but reversibly, suppressed the fraction of S-phase cells. Dexamethasone also reversibly repressed expression of thymidylate synthase and dihydrofolate reductase, which are primary targets of pemetrexed but are also quintessential S-phase enzymes as well as the S-phase-dependent expression of thymidine kinase 1. Dexamethasone also decreased expression of the major pemetrexed transporters, the reduced folate carrier and the proton coupled folate transporter. Only cells expressing relatively high GRα showed these dexamethasone effects, regardless of p53 status. In cells expressing low GRα, the dexamethasone response was rescued by ectopic GRα. Further, depletion of p53 did not attenuate the dexamethasone effects. The presence of dexamethasone during pemetrexed treatment protected against pemetrexed cytotoxicity in only the dexamethasone responsive cells. CONCLUSIONS: The results predict that in nonsquamous NSCLC tumors, reversible S-phase suppression by dexamethasone, possibly combined with a reduction in the drug transporters, attenuates responsiveness to pemetrexed and that GR status is a principal determinant of tumor variability of this response.