New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types.

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg- ) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Spatial Gene-Expression Profiling Unveils Immuno-oncogenic Programs of NF1-Associated Peripheral Nerve Sheath Tumor Progression.

PURPOSE: Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors (PNST) associated with neurofibromatosis type 1 (NF1). Despite similar histologic appearance, these neoplasms exhibit diverse evolutionary trajectories, with a subset progressing to malignant peripheral nerve sheath tumor (MPNST), the leading cause of premature death in individuals with NF1. Malignant transformation of PNF often occurs through the development of atypical neurofibroma (ANF) precursor lesions characterized by distinct histopathologic features and CDKN2A copy-number loss. Although genomic studies have uncovered key driver events promoting tumor progression, the transcriptional changes preceding malignant transformation remain poorly defined. EXPERIMENTAL DESIGN: Here we resolve gene-expression profiles in PNST across the neurofibroma-to-MPNST continuum in NF1 patients and mouse models, revealing early molecular features associated with neurofibroma evolution and transformation. RESULTS: Our findings demonstrate that ANF exhibit enhanced signatures of antigen presentation and immune response, which are suppressed as malignant transformation ensues. MPNST further displayed deregulated survival and mitotic fidelity pathways, and targeting key mediators of these pathways, CENPF and BIRC5, disrupted the growth and viability of human MPNST cell lines and primary murine Nf1-Cdkn2a-mutant Schwann cell precursors. Finally, neurofibromas contiguous with MPNST manifested distinct alterations in core oncogenic and immune surveillance programs, suggesting that early molecular events driving disease progression may precede histopathologic evidence of malignancy. CONCLUSIONS: If validated prospectively in future studies, these signatures may serve as molecular diagnostic tools to augment conventional histopathologic diagnosis by identifying neurofibromas at high risk of undergoing malignant transformation, facilitating risk-adapted care.

Exploring the Tumor-Suppressing Potential of PSCA in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates. We explored an innovative therapeutic approach by leveraging prognostic oncogenic markers. Instead of inhibiting these marker genes, we harnessed their tumor-modifying potential in the extracellular domain. Surprisingly, many of the proteins highly expressed in PDAC, which is linked to poor survival, exhibited tumor-suppressing qualities in the extracellular environment. For instance, prostate stem cell antigens (PSCA), associated with reduced survival, acted as tumor suppressors when introduced extracellularly. We performed in vitro assays to assess the proliferation and migration and evaluated the tumor-modifying capacity of extracellular factors from peripheral blood mononuclear cells (PBMCs) in PDAC tissues. Molecular docking analysis, immunoprecipitation, Western blotting, and RNA interference were employed to study the regulatory mechanism. Extracellular PSCA recombinant protein notably curtailed the viability, motility, and transwell invasion of PDAC cells. Its anti-PDAC effects were partially mediated by Mesothelin (MSLN), another highly expressed tumor-associated antigen in PDAC. The anti-tumor effects of extracellular PSCA complemented those of chemotherapeutic agents like Irinotecan, 5-Fluorouracil, and Oxaliplatin. PSCA expression increased in a conditioned medium derived from PBMCs and T lymphocytes. This study unveils the paradoxical anti-PDAC potential of PSCA, hinting at the dual roles of oncoproteins like PSCA in PDAC suppression.

Anticancer Peptides Derived from Aldolase A and Induced Tumor-Suppressing Cells Inhibit Pancreatic Ductal Adenocarcinoma Cells.

PDAC (pancreatic ductal adenocarcinoma) is a highly aggressive malignant tumor. We have previously developed induced tumor-suppressing cells (iTSCs) that secrete a group of tumor-suppressing proteins. Here, we examined a unique procedure to identify anticancer peptides (ACPs), using trypsin-digested iTSCs-derived protein fragments. Among the 10 ACP candidates, P04 (IGEHTPSALAIMENANVLAR) presented the most efficient anti-PDAC activities. P04 was derived from aldolase A (ALDOA), a glycolytic enzyme. Extracellular ALDOA, as well as P04, was predicted to interact with epidermal growth factor receptor (EGFR), and P04 downregulated oncoproteins such as Snail and Src. Importantly, P04 has no inhibitory effect on mesenchymal stem cells (MSCs). We also generated iTSCs by overexpressing ALDOA in MSCs and peripheral blood mononuclear cells (PBMCs). iTSC-derived conditioned medium (CM) inhibited the progression of PDAC cells as well as PDAC tissue fragments. The inhibitory effect of P04 was additive to that of CM and chemotherapeutic drugs such as 5-Flu and gemcitabine. Notably, applying mechanical vibration to PBMCs elevated ALDOA and converted PBMCs into iTSCs. Collectively, this study presented a unique procedure for selecting anticancer P04 from ALDOA in an iTSCs-derived proteome for the treatment of PDAC.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Pancreatic cancer is a major cause of cancer-related deaths worldwide, with only 12% of patients surviving for five years after diagnosis. Individuals generally experience few symptoms of the disease in the early stages and are often diagnosed once the cancer has already spread to other parts of the body. By this point, options for treatment are limited. A molecule known as triptolide has been shown to kill breast, lung, pancreatic and other types of cancer cells. However, triptolide is toxic to humans and other animals, making it unsuitable for use in patients. One way to make drugs safer without compromising their beneficial effects is to modify their molecular structure. By formulating triptolide into an emulsion ­ a mixture of liquids allowing it to dissolve ­ Tian, Zhang et al. synthesized a new analogue called CK21. Experiments showed that CK21 inhibited the growth of human pancreatic cancer cells grown in a laboratory including cells grown in artificial organs similar to the pancreas, known as pancreatic tumor organoids. Furthermore, CK21 killed large tumors in mice pancreases with very few side effects, suggesting the structural modification of triptolide increased safety of the drug. To better understand how CK21 works, Tian, Zhang et al. examined the genes that were induced in the pancreatic tumor organoids at various time points after treatment with the drug. This revealed that CK21 switched off genes involved in the NF-κB cell signaling pathway, which regulates how cells grow and respond to stress. In turn, it triggered programmed cell death, killing the tumor cells in a controlled manner. The findings suggest that CK21 could be a promising candidate for treating pancreatic cancer. In the future, clinical trials will be required to establish whether CK21 is a safe and effective therapy for humans.

Cancer-associated fibroblasts influence Wnt/PCP signaling in gastric cancer cells by cytoneme-based dissemination of ROR2.

Cancer-associated fibroblasts (CAFs) are a crucial component in the tumor microenvironment influencing cancer progression. Besides shaping the extracellular matrix, these fibroblasts provide signaling factors to facilitate tumor survival and alter tumor behavior. In gastric cancer, one crucial signaling pathway influencing invasion and metastasis is the Wnt/Planar Cell Polarity (PCP) signaling. The crucial PCP ligand in this context is WNT5A, which is produced by the CAFs, and gastric cancer cells react upon this signal by enhanced polarized migration. Why gastric cancer cells respond to this signal is still unclear, as their expression level for the central WNT5A receptor, ROR2, is very low. Here, we show that CAFs display long and branched filopodia that form an extensive, complex network engulfing gastric cancer cells, such as the gastric cancer cell line AGS. CAFs have a significantly higher expression level of ROR2 than normal gastric fibroblasts and AGS cells. By high-resolution imaging, we observe a direct transfer of fluorescently tagged ROR2 from CAF to AGS cells by signaling filopodia, known as cytonemes. Surprisingly, we find that the transferred ROR2 complexes can activate Wnt/JNK signaling in AGS cells. Consistently, blockage of ROR2 function in the CAFs leads to reduced paracrine Wnt/JNK signaling, cell polarization, and migration of the receiving AGS cells. Complementary, enhanced migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These findings demonstrate a fresh role for cytoneme-mediated signaling in the tumor microenvironment. Cytonemes convey Wnt receptors from CAFs to gastric cancer cells, allowing them to respond to Wnt/PCP signals.

The inhibition of pancreatic cancer progression by K-Ras-overexpressing mesenchymal stem cell-derived secretomes.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival. To explore an uncharted function of K-Ras proto-oncogene, K-Ras was activated in mesenchymal stem cells (MSCs) and the effects of MSC conditioned medium (CM) on PDAC were examined. Overexpression of K-Ras elevated PI3K signaling in MSCs, and K-Ras/PI3K-activated MSC-derived CM reduced the proliferation and migration of tumor cells, as well as the growth of ex vivo freshly isolated human PDAC cultures. CM's anti-tumor capability was additive with Gemcitabine, a commonly used chemotherapeutic drug in the treatment of PDAC. The systemic administration of CM in a mouse model suppressed the colonization of PDAC in the lung. MSC CM was enriched with Moesin (MSN), which acted as an extracellular tumor-suppressing protein by interacting with CD44. Tumor-suppressive CM was also generated by PKA-activated peripheral blood mononuclear cells. Collectively, this study demonstrated that MSC CM can be engineered to act as a tumor-suppressive agent by activating K-Ras and PI3K, and the MSN-CD44 regulatory axis is in part responsible for this potential unconventional option in the treatment of PDAC.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue.

Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays in vitro. Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using in silico prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth in vivo. Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.

Viscoelastic hydrogels for interrogating pancreatic cancer-stromal cell interactions.

The tumor microenvironment (TME) is known to direct cancer cell growth, migration, invasion into the matrix and distant tissues, and to confer drug resistance in cancer cells. While multiple aspects of TME have been studied using in vitro, ex vivo, and in vivo tumor models and engineering tools, the influence of matrix viscoelasticity on pancreatic cancer cells and its associated TME remained largely unexplored. In this contribution, we synthesized a new biomimetic hydrogel with tunable matrix stiffness and stress-relaxation for evaluating the effect of matrix viscoelasticity on pancreatic cancer cell (PCC) behaviors in vitro. Using three simple monomers and Reverse-Addition Fragmentation Chain-Transfer (RAFT) polymerization, we synthesized a new class of phenylboronic acid containing polymers (e.g., poly (OEGA-s-HEAA-s-APBA) or PEHA). Norbornene group was conjugated to HEAA on PEHA via carbic anhydride, affording a new NB and BA dually modified polymer - PEHNBA amenable for orthogonal thiol-norbornene photopolymerization and boronate ester diol complexation. The former provided tunable matrix elasticity, while the latter gave rise to matrix stress-relaxation (or viscoelasticity). The new PEHNBA polymers were shown to be highly cytocompatible for in situ encapsulation of PCCs and cancer-associated fibroblasts (CAFs). Furthermore, we demonstrated that hydrogels with high stress-relaxation promoted spreading of CAFs, which in turns promoted PCC proliferation and spreading in the viscoelastic matrix. Compared with elastic matrix, viscoelastic gels upregulated the secretion of soluble proteins known to promote epithelial-mesenchymal transition (EMT). This study demonstrated the crucial influence of matrix viscoelasticity on pancreatic cancer cell fate and provided an engineered viscoelastic matrix for future studies and applications related to TME.

Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that is often detected at an advanced stage. Earlier diagnosis of PDAC is key to reducing mortality. Circulating biomarkers such as microRNAs are gaining interest, but existing technologies require large sample volumes, amplification steps, extensive biofluid processing, lack sensitivity, and are low-throughput. Here, we present an advanced nanoplasmonic sensor for the highly sensitive, amplification-free detection and quantification of microRNAs (microRNA-10b, microRNA-let7a) from unprocessed plasma microsamples. The sensor construct utilizes uniquely designed -ssDNA receptors attached to gold triangular nanoprisms, which display unique localized surface plasmon resonance (LSPR) properties, in a multiwell plate format. The formation of -ssDNA/microRNA duplex controls the nanostructure-biomolecule interfacial electronic interactions to promote the charge transfer/exciton delocalization processes and enhance the LSPR responses to achieve attomolar (10-18 M) limit of detection (LOD) in human plasma. This improve LOD allows the fabrication of a high-throughput assay in a 384-well plate format. The performance of nanoplasmonic sensors for microRNA detection was further assessed by comparing with the qRT-PCR assay of 15 PDAC patient plasma samples that shows a positive correlation between these two assays with the Pearson correlation coefficient value >0.86. Evaluation of >170 clinical samples reveals that oncogenic microRNA-10b and tumor suppressor microRNA-let7a levels can individually differentiate PDAC from chronic pancreatitis and normal controls with >94% sensitivity and >94% specificity at a 95% confidence interval (CI). Furthermore, combining both oncogenic and tumor suppressor microRNA levels significantly improves differentiation of PDAC stages I and II versus III and IV with >91% and 87% sensitivity and specificity, respectively, in comparison to the sensitivity and specificity values for individual microRNAs. Moreover, we show that the level of microRNAs varies substantially in pre- and post-surgery PDAC patients (n = 75). Taken together, this ultrasensitive nanoplasmonic sensor with excellent sensitivity and specificity is capable of assaying multiple biomarkers simultaneously and may facilitate early detection of PDAC to improve patient care.

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

Refining colorectal cancer classification and clinical stratification through a single-cell atlas.

BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.

RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered Kras G12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the Kras G12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

Hypoxia signaling: Challenges and opportunities for cancer therapy.

Hypoxia is arguably the first recognized cancer microenvironment hallmark and affects virtually all cellular populations present in tumors. During the past decades the complex adaptive cellular responses to oxygen deprivation have been largely elucidated, raising hope for new anti cancer agents. Despite undeniable preclinical progress, therapeutic targeting of tumor hypoxia is yet to transition from bench to bedside. This review focuses on new pharmacological agents that exploit tumor hypoxia or interfere with hypoxia signaling and discusses strategies to maximize their therapeutic impact.

Clinical and Preclinical Outcomes of Combining Targeted Therapy With Radiotherapy.

In the era of precision medicine, radiation medicine is currently focused on the precise delivery of highly conformal radiation treatments. However, the tremendous developments in targeted therapy are yet to fulfill their full promise and arguably have the potential to dramatically enhance the radiation therapeutic ratio. The increased ability to molecularly profile tumors both at diagnosis and at relapse and the co-incident progress in the field of radiogenomics could potentially pave the way for a more personalized approach to radiation treatment in contrast to the current ''one size fits all'' paradigm. Few clinical trials to date have shown an improved clinical outcome when combining targeted agents with radiation therapy, however, most have failed to show benefit, which is arguably due to limited preclinical data. Several key molecular pathways could theoretically enhance therapeutic effect of radiation when rationally targeted either by directly enhancing tumor cell kill or indirectly through the abscopal effect of radiation when combined with novel immunotherapies. The timing of combining molecular targeted therapy with radiation is also important to determine and could greatly affect the outcome depending on which pathway is being inhibited.

Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions.

Acute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O2 )-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O2 -deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O2 conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O2 -deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.