Tumor and Peritoneum-Associated Macrophage Gene Signature as a Novel Molecular Biomarker in Gastric Cancer.

A spectrum of immune states resulting from tumor resident macrophages and T-lymphocytes in the solid tumor microenvironment correlates with patient outcomes. We hypothesized that in gastric cancer (GC), macrophages in a polarized immunosuppressive transcriptional state would be prognostic of poor survival. We derived transcriptomic signatures for M2 (M2TS, MRC1; MS4A4A; CD36; CCL13; CCL18; CCL23; SLC38A6; FGL2; FN1; MAF) and M1 (M1TS, CCR7; IL2RA; CXCL11; CCL19; CXCL10; PLA1A; PTX3) macrophages, and cytolytic T-lymphocytes (CTLTS, GZMA; GZMB; GZMH; GZMM; PRF1). Primary GC in a TCGA stomach cancer dataset was evaluated for signature expressions, and a log-rank test determined overall survival (OS) and the disease-free interval (DFI). In 341 TCGA GC entries, high M2TS expression was associated with histological types and later stages. Low M2TS expression was associated with significantly better 5-year OS and DFI. We validated M2TS in prospectively collected peritoneal fluid of a GC patient cohort (n = 28). Single-cell RNA sequencing was used for signature expression in CD68+CD163+ cells and the log-rank test compared OS. GC patients with high M2TS in CD68+CD163+ cells in their peritoneal fluid had significantly worse OS than those with low expression. Multivariate analyses confirmed M2TS was significantly and independently associated with survival. As an independent predictor of poor survival, M2TS may be prognostic in primary tumors and peritoneal fluid of GC patients.

First-in-Human Phase I Study of Minnelide in Patients With Advanced Gastrointestinal Cancers: Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity.

BACKGROUND: Minnelide is a water-soluble prodrug of triptolide. Triptolide is an anticancer agent that targets cancer resistance through several mechanisms. Minnelide was evaluated in a phase I study in patients with advanced GI carcinomas to establish the safety, pharmacodynamic, antitumor activity, and recommended phase II dose (RP2D). PATIENTS AND METHODS: Patients with refractory GI carcinoma and with measurable disease on CT scan were eligible. The study used a 3 + 3 dose-escalation scheme. Due to neutropenia toxicity, 2 dosing schedules were evaluated to determine the RP2D for future studies. Response was assessed using RECIST 1.1 and Choi criteria. Minnelide and triptolide PK were evaluated. Patients who completed the first 28-day treatment cycle without DLTs continued treatment until disease progression or unacceptable toxicity. RESULTS: Forty-five patients were enrolled (23 pancreatic cancer, 10 colorectal, and the remaining 9 had other GI tumors); 42 patients received at least one dose of Minnelide. Grade ≥ 3 toxicities occurred in 69% of patients, most common neutropenia (38%). 2 patients with severe cerebellar toxicity who had a 2-fold higher triptolide concentration than other participants. ORR was 4%; the disease control rate (DCR) was 54% (15/28). Choi criteria demonstrated a decrease in average tumor density in 57% (16/28) patients. CONCLUSIONS: This first-in-human, phase I clinical study identified a dose and schedule of Minnelide in patients with refractory GI cancers. The primary toxicity experienced was hematologic. Evidence of efficacy of Minnelide treatment in this group of patients was observed. The DCR ranged from ~2 to 6 months in 14/28 (50%) of evaluable patients. Studies in monotherapy and combination treatments are underway.

A Circulating Panel of circRNA Biomarkers for the Noninvasive and Early Detection of Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies. Delayed manifestation of symptoms and lack of specific diagnostic markers lead patients being diagnosed with PDAC at advanced stages. This study aimed to develop a circular RNA (circRNA)-based biomarker panel to facilitate noninvasive and early detection of PDAC. METHODS: A systematic genome-wide discovery of circRNAs overexpressed in patients with PDAC was conducted. Subsequently, validation of the candidate markers in the primary tumors from patients with PDAC was performed, followed by their translation into a plasma-based liquid biopsy assay by analyzing 2 independent clinical cohorts of patients with PDAC and nondisease controls. The performance of the circRNA panel was assessed in conjunction with the plasma levels of cancer antigen 19-9 for the early detection of PDAC. RESULTS: Initially, a panel of 10 circRNA candidates was identified during the discovery phase. Subsequently, the panel was reduced to 5 circRNAs in the liquid biopsy-based assay, which robustly identified patients with PDAC and distinguished between early-stage (stage I/II) and late-stage (stage III/IV) disease. The areas under the curve of this diagnostic panel for the detection of early-stage PDAC were 0.83 and 0.81 in the training and validation cohorts, respectively. Moreover, when this panel was combined with cancer antigen 19-9 levels, the diagnostic performance for identifying patients with PDAC improved remarkably (area under the curve, 0.94) for patients in the validation cohort. Furthermore, the circRNA panel could also efficiently identify patients with PDAC (area under the curve, 0.85) who were otherwise deemed clinically cancer antigen 19-9-negative (<37 U/mL). CONCLUSIONS: A circRNA-based biomarker panel with a robust noninvasive diagnostic potential for identifying patients with early-stage PDAC was developed.

Inhibiting stromal Class I HDACs curbs pancreatic cancer progression.

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Advanced glycation end product (AGE) targeting antibody SIWA318H is efficacious in preclinical models for pancreatic cancer.

SIWA318H is a novel monoclonal antibody that selectively targets an advanced glycation end product biomarker found in damaged/dysfunctional cells exhibiting (a) aerobic glycolysis, and (b) oxidative stress. Cells with this biomarker are dysfunctional and are associated with stresses and/or damages relating to aging, cancer and other disease processes. In this study, we evaluated the biological effects and antitumor activity of SIWA318H in preclinical models for pancreatic cancer. SIWA318H binds to pancreatic cancer cells and cancer-associated fibroblasts, as well as tumor xenografts derived from pancreatic cancer patients. Furthermore, SIWA318H induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) against pancreatic cancer cells. In a humanized CD34+ NSG mouse xenograft model for pancreatic cancer, tumors in mice treated with SIWA318H grew significantly slower compared to those in control mice (p < 0.001). After 3 weeks of treatment with SIWA318H, the tumor growth was suppressed by 68.8% and 61.5% for the high and low dose regimens, respectively, when compared to the isotype antibody control (ANOVA p < 0.002). Moreover, a significant increase in complete remission (CR) rate was observed in mice receiving the high dose (60%, p < 0.04) or low dose (77.8%, p < 0.02) of SIWA318H treatment compared with control mice (6.7%). Immunohistochemical analyses of the tumor tissues showed a significant decrease in senescent cells in the tumor microenvironment of SIWA318H treated mice compared to that of control treated mice (p < 0.05). These results provide compelling evidence that SIWA318H is a promising novel therapeutic against pancreatic cancer.

Single-cell mapping identifies MSI+ cells as a common origin for diverse subtypes of pancreatic cancer.

Identifying the cells from which cancers arise is critical for understanding the molecular underpinnings of tumor evolution. To determine whether stem/progenitor cells can serve as cells of origin, we created a Msi2-CreERT2 knock-in mouse. When crossed to CAG-LSL-MycT58A mice, Msi2-CreERT2 mice developed multiple pancreatic cancer subtypes: ductal, acinar, adenosquamous, and rare anaplastic tumors. Combining single-cell genomics with computational analysis of developmental states and lineage trajectories, we demonstrate that MYC preferentially triggers transformation of the most immature MSI2+ pancreas cells into multi-lineage pre-cancer cells. These pre-cancer cells subsequently diverge to establish pancreatic cancer subtypes by activating distinct transcriptional programs and large-scale genomic changes, and enforced expression of specific signals like Ras can redirect subtype specification. This study shows that multiple pancreatic cancer subtypes can arise from a common pool of MSI2+ cells and provides a powerful model to understand and control the programs that shape divergent fates in pancreatic cancer.

Inhibiting Stromal Class I HDACs Curbs Pancreatic Cancer Progression.

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Tumor Response to Stroma-Modifying Therapy: Magnetic Resonance Imaging Findings in Early-Phase Clinical Trials of Pegvorhyaluronidase alpha (PEGPH20).

Pre-clinical and clinical studies have shown that PEGPH20 depletes intratumoral hyaluronic acid (HA), which is linked to high interstitial fluid pressures and poor distribution of chemotherapies. 29 patients with metastatic advanced solid tumors received quantitative magnetic resonance imaging (qMRI) in 3 prospective clinical trials of PEGPH20, HALO-109-101 (NCT00834704), HALO-109-102 (NCT01170897), and HALO-109-201 (NCT01453153). Apparent Diffusion Coefficient of water (ADC), T1, ktrans, vp, ve, and iAUC maps were computed from qMRI acquired at baseline and ≥ 1 time point post-PEGPH20. Tumor ADC and T1 decreased, while iAUC, ktrans, vp, and ve increased, on day 1 post-PEGPH20 relative to baseline values. This is consistent with HA depletion leading to a decrease in tumor water content and an increase in perfusion, permeability, extracellular matrix space, and vascularity. Baseline parameter values that were predictive of pharmacodynamic responses were: ADC > 1.46×10-3 mm2/s (Balanced Accuracy (BA) = 72%, p < 0.01), T1 > 0.54s (BA = 82%, p < 0.01), iAUC < 9.2 mM-s (BA = 76%, p < 0.05), ktrans<0.07min-1 (BA = 72%, p = 0.2), ve<0.17 (BA = 68%, p < 0.01), and vp<0.02 (BA = 60%, p < 0.01). Further, ve<0.39 at baseline was moderately predictive of response in any parameter (BA = 65.6%, p < 0.01 averaged across patients). These qMRI biomarkers are potentially useful for guiding patient pre-selection and post-treatment follow-up in future clinical studies of PEGPH20 and other tumor stroma-modifying anti-cancer therapies.

A super-enhancer-regulated RNA-binding protein cascade drives pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.

Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Can Imaging Using Radiomics and Fat Fraction Analysis Detect Early Tissue Changes on Historical CT Scans in the Regions of the Pancreas Gland That Subsequently Develop Adenocarcinoma?

Despite a growing number of effective therapeutic options for patients with pancreatic adenocarcinoma, the prognosis remains dismal mostly due to the late-stage presentation and spread of the cancer to other organs. Because a genomic analysis of pancreas tissue revealed that it may take years, if not decades, for pancreatic cancer to develop, we performed radiomics and fat fraction analysis on contrast-enhanced CT (CECT) scans of patients with historical scans showing no evidence of cancer but who subsequently went on to develop pancreas cancer years later, in an attempt to identify specific imaging features of the normal pancreas that may portend the subsequent development of the cancer. In this IRB-exempt, retrospective, single institution study, CECT chest, abdomen, and pelvis (CAP) scans of 22 patients who had evaluable historical imaging data were analyzed. The images from the "healthy" pancreas were obtained between 3.8 and 13.9 years before the diagnosis of pancreas cancer was established. Afterwards, the images were used to divide and draw seven regions of interest (ROIs) around the pancreas (uncinate, head, neck-genu, body (proximal, middle, and distal) and tail). Radiomic analysis on these pancreatic ROIs consisted of first order quantitative texture analysis features such as kurtosis, skewness, and fat quantification. Of all the variables tested, fat fraction in the pancreas tail (p = 0.029) and asymmetry of the histogram frequency curve (skewness) of pancreas tissue (p = 0.038) were identified as the most important imaging signatures for subsequent cancer development. Changes in the texture of the pancreas as measured on the CECT of patients who developed pancreas cancer years later could be identified, confirming the utility of radiomics-based imaging as a potential predictor of oncologic outcomes. Such findings may be potentially useful in the future to screen patients for pancreatic cancer, thereby helping detect pancreas cancer at an early stage and improving survival.

Tumor Growth Rate Informs Treatment Efficacy in Metastatic Pancreatic Adenocarcinoma: Application of a Growth and Regression Model to Pivotal Trial and Real-World Data.

BACKGROUND: Methods for screening agents earlier in development and strategies for conducting smaller randomized controlled trials (RCTs) are needed. METHODS: We retrospectively applied a tumor growth model to estimate the rates of growth of pancreatic cancer using radiographic tumor measurements or serum CA 19-9 values from 3033 patients with stages III-IV PDAC who were enrolled in 8 clinical trials or were included in 2 large real-world data sets. RESULTS: g correlated inversely with OS and was consistently lower in the experimental arms than in the control arms of RCTs. At the individual patient level, g was significantly faster for lesions metastatic to the liver relative to those localized to the pancreas. Regardless of regimen, g increased toward the end of therapy, often by over 3-fold. CONCLUSIONS: Growth rates of PDAC can be determined using radiographic tumor measurement and CA 19-9 values. g is inversely associated with OS and can differentiate therapies within the same trial and across trials. g can also be used to characterize changes in the behavior of an individual's PDAC, such as differences in the growth rate of lesions based on metastatic site, and the emergence of chemoresistance. We provide examples of how g can be used to benchmark phase II and III clinical data to a virtual reference arm to inform go/no go decisions and consider novel trial designs to optimize and accelerate drug development.

Effect of acoustic cluster therapy (ACT®) combined with chemotherapy in a patient-derived xenograft mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinomas respond poorly to chemotherapy, in part due to the dense tumor stroma that hinders drug delivery. Ultrasound (US) in combination with microbubbles has previously shown promise as a means to improve drug delivery, and the therapeutic efficacy of ultrasound-mediated drug delivery is currently being evaluated in multiple clinical trials. However, most of these utilize echogenic contrast agents engineered for imaging, which might not be optimal compared to specialized formulations tailored for drug delivery. In this study, we evaluated the in vivo efficacy of phase-shifting microbubble-microdroplet clusters that, upon insonation, form bubbles in the size range of 20-30 µm. We developed a patient-derived xenograft model of pancreatic cancer implanted in mice that largely retained the stromal content of the originating tumor and compared tumor growth in mice given chemotherapeutics (nab-paclitaxel plus gemcitabine or liposomal irinotecan) with mice given the same chemotherapeutics in addition to ultrasound and acoustic cluster therapy. We found that acoustic cluster therapy significantly improved the effect of both chemotherapeutic regimens and resulted in 7.2 times higher odds of complete remission of the tumor compared to the chemotherapeutics alone.

Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

An Exosome-based Transcriptomic Signature for Noninvasive, Early Detection of Patients With Pancreatic Ductal Adenocarcinoma: A Multicenter Cohort Study.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) incidence is rising worldwide, and most patients present with an unresectable disease at initial diagnosis. Measurement of carbohydrate antigen 19-9 (CA19-9) levels lacks adequate sensitivity and specificity for early detection; hence, there is an unmet need to develop alternate molecular diagnostic biomarkers for PDAC. Emerging evidence suggests that tumor-derived exosomal cargo, particularly micro RNAs (miRNAs), offer an attractive platform for the development of cancer-specific biomarkers. Herein, genomewide profiling in blood specimens was performed to develop an exosome-based transcriptomic signature for noninvasive and early detection of PDAC. METHODS: Small RNA sequencing was undertaken in a cohort of 44 patients with an early-stage PDAC and 57 nondisease controls. Using machine-learning algorithms, a panel of cell-free (cf) and exosomal (exo) miRNAs were prioritized that discriminated patients with PDAC from control subjects. Subsequently, the performance of the biomarkers was trained and validated in independent cohorts (n = 191) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. RESULTS: The sequencing analysis initially identified a panel of 30 overexpressed miRNAs in PDAC. Subsequently using qRT-PCR assays, the panel was reduced to 13 markers (5 cf- and 8 exo-miRNAs), which successfully identified patients with all stages of PDAC (area under the curve [AUC] = 0.98 training cohort; AUC = 0.93 validation cohort); but more importantly, was equally robust for the identification of early-stage PDAC (stages I and II; AUC = 0.93). Furthermore, this transcriptomic signature successfully identified CA19-9 negative cases (<37 U/mL; AUC = 0.96), when analyzed in combination with CA19-9 levels, significantly improved the overall diagnostic accuracy (AUC = 0.99 vs AUC = 0.86 for CA19-9 alone). CONCLUSIONS: In this study, an exosome-based liquid biopsy signature for the noninvasive and robust detection of patients with PDAC was developed.

A phase II trial of the super-enhancer inhibitor Minnelide™ in advanced refractory adenosquamous carcinoma of the pancreas.

Adenosquamous carcinoma of the pancreas (ASCP) is a very rare and highly aggressive variant of pancreatic ductal adenocarcinoma, accounting for 0.5-4% of all pancreatic cancer cases in the USA. Current data indicate that epigenetic changes and MYC overexpression lead to squamous transdifferentiation of pancreatic tumor cells and development of ASCP. Minnelide™, an oral anti-super-enhancer drug that inhibits MYC expression in preclinical models of ASCP, has demonstrated safety in a phase I study. We describe the design for a phase II, open-label, single-arm trial of Minnelide in patients with advanced refractory ASCP.

Adenosquamous carcinoma of the pancreas (ASCP) is a rare and highly aggressive variant of pancreatic cancer, with limited treatment options. Changes in activation of DNA elements called super-enhancers drive the growth of ASCP. Minnelide™ is an oral drug that blocks the super-enhancer network and is safe to give to patients with advanced cancer. This trial is designed to determine whether Minnelide can shrink tumors in patients with ASCP who have already received at least one previous treatment for their cancer.  Clinical Trial Registration: NCT04896073 (ClinicalTrials.gov).

Ketogenic diet and chemotherapy combine to disrupt pancreatic cancer metabolism and growth.

Background: Ketogenic diet is a potential means of augmenting cancer therapy. Here, we explore ketone body metabolism and its interplay with chemotherapy in pancreatic cancer. Methods: Metabolism and therapeutic responses of murine pancreatic cancer were studied using KPC primary tumors and tumor chunk allografts. Mice on standard high-carbohydrate diet or ketogenic diet were treated with cytotoxic chemotherapy (nab-paclitaxel, gemcitabine, cisplatin). Metabolic activity was monitored with metabolomics and isotope tracing, including 2H- and 13C-tracers, liquid chromatography-mass spectrometry, and imaging mass spectrometry. Findings: Ketone bodies are unidirectionally oxidized to make NADH. This stands in contrast to the carbohydrate-derived carboxylic acids lactate and pyruvate, which rapidly interconvert, buffering NADH/NAD. In murine pancreatic tumors, ketogenic diet decreases glucose's concentration and tricarboxylic acid cycle contribution, enhances 3-hydroxybutyrate's concentration and tricarboxylic acid contribution, and modestly elevates NADH, but does not impact tumor growth. In contrast, the combination of ketogenic diet and cytotoxic chemotherapy substantially raises tumor NADH and synergistically suppresses tumor growth, tripling the survival benefits of chemotherapy alone. Chemotherapy and ketogenic diet also synergize in immune-deficient mice, although long-term growth suppression was only observed in mice with an intact immune system. Conclusions: Ketogenic diet sensitizes murine pancreatic cancer tumors to cytotoxic chemotherapy. Based on these data, we have initiated a randomized clinical trial of chemotherapy with standard versus ketogenic diet for patients with metastatic pancreatic cancer (NCT04631445).

Mechanisms of GZ17-6.02 resistance.

OBJECTIVES: The drug GZ17-6.02 is undergoing phase I in solid tumor patients (NCT03775525). The present studies initially determined the impact of prolonged exposure of colorectal tumors to GZ17-6.02, and to determine whether GZ17-6.02 enhanced the efficacy of an anti-PD1 antibody. Subsequently, studies defined the evolutionary resistance mechanisms in tumor cells previously exposed to GZ17-6.02. METHODS: IACUC-approved animal studies were performed. In cell immunoblotting, cell transfections and trypan blue death assays were performed. RESULTS: Prolonged exposure of colorectal tumors to GZ17-6.02 enhanced the efficacy of 5-fluorouracil and of an anti-PD1 antibody, significantly prolonging animal survival. Tumor cells previously exposed to GZ17-6.02 in vivo had elevated their expression of ERBB2 and ERBB3, and increased phosphorylation of ERBB1, ERBB3, PDGFRß, AKT T308, ERK1/2, p70 S6K T389, STAT5 Y694 and c-SRC Y416. The phosphorylation of c-SRC Y527 declined. The efficacy of ERBB receptor inhibitors at killing these resistant tumor cells was unaltered by prior GZ17-6.02 exposure whereas the efficacy of multi-kinase/PDGFRß inhibitors was significantly reduced. Treatment of colon cancer cells with GZ17-6.02 rapidly reduced the levels of multiple HDAC proteins and altered their subcellular localization. Isolates from resistant tumors expressed less CD95 and FAS-L. HDAC inhibitors enhanced CD95 and FAS-L levels in the resistant cells via activation of NFκB and HDAC inhibitors restored the efficacy of GZ17-6.02 to near control levels. CONCLUSIONS: Our findings demonstrate that GZ17-6.02 has the potential to be developed as a colon cancer therapeutic and that resistance to the drug can be partially reversed by HDAC inhibitors.