Ongoing Studies Research Articles

Pirtobrutinib: A New and Distinctive Treatment Option for B-Cell Malignancies.

The objective was to evaluate the efficacy/safety of pirtobrutinib in the treatment of B-cell malignancies and distinguish it from other available Bruton's tyrosine kinase (BTK) inhibitors. A literature search of PubMed (January 2021 through November 2023) and Clinicaltrials.gov was conducted using terms pirtobrutinib, Jaypirca, and LOXO 305. Licensing trials of available BTK inhibitors were also reviewed. Relevant English-language clinical trials were evaluated. Pirtobrutinib was approved by the US Food and Drug Administration for the treatment of relapsed/refractory mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) based largely on a phase 1/2 study in B-cell malignancies. Pirtobrutinib demonstrated a 73% overall response rate (ORR) in the CLL population and 58% in MCL. Pirtobrutinib has activity in patients resistant to earlier-generation, covalent BTK inhibitors. In fact, the ORRs were similar in BTK-pretreated and naïve patients. Adverse effects include fatigue, diarrhea, bleeding, and infection. Atrial fibrillation, a class effect of BTK inhibitors, may be less common with pirtobrutinib. Compared with earlier-generation BTK inhibitors, pirtobrutinib is more selective for BTK and binds noncovalently to the receptor. Ongoing studies are evaluating pirtobrutinib's use in multiple B-cell malignancies and comparing it with other BTK inhibitors. The characteristics of pirtobrutinib render it useful in the treatment of B-cell malignancies no longer responding to a previous BTK inhibitor, and results from ongoing clinical trials may support future expanded use.

Abstract C103: Differential acquisition of extracellular lipid correlates with pancreatic cancer subtype and metastatic tropism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease, and the third leading cause of cancer-related death with a 5-year overall survival rate of less than 5%. Recent studies have identified two major subtypes of pancreatic cancer, basal and classical, that are predictive of patient prognosis, with basal tumors corresponding with more aggressive disease. While transcriptional signatures define basal and classical tumors and cell lines, little is known regarding the metabolic vulnerabilities linked to the basal versus classical state. Using unbiased computational interrogation to uncover metabolic genes correlating with known markers of classical PDAC, we identified high expression of Proprotein convertase subtilisin/kexin type-9 (PCSK9) – a negative regulator of receptor mediated uptake of cholesterol containing low-density lipoprotein (LDL) – as correlating with several established markers of classical PDAC. In contrast, PCSK9 expression is suppressed in Basal PDAC. Accordingly, basal PDAC uptake high levels of LDL, are sensitive to LDL depletion and show higher dependence on cholesterol uptake, relative to classical PDAC. As lipoproteins are produced in the liver, we hypothesized that basal PDAC cells may more efficiently seed and grow within the liver. Consistent with this hypothesis, low PCSK9 expression is correlated with liver metastasis in mouse models of PDAC and patient PDAC specimens. Ongoing studies will interrogate the role of PCSK9 and cholesterol uptake and utilization in metastatic tropism, and uncover unique features and vulnerabilities of the most aggressive variant of PDAC. Citation Format: Gilles Rademaker, Grace A. Hernandez, Longhui Qiu, Kwun W. Wen, Aakriti Jain, Grace A. Kim, Roberto Zoncu, Rushik M. Perera. Differential acquisition of extracellular lipid correlates with pancreatic cancer subtype and metastatic tropism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C103.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

Abstract C048: Obesity transforms the gut microbiome to invoke a cancer permissive state

Abstract Obesity – an established, modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC) – has increased in prevalence worldwide yet the precise mechanisms underlying its propensity to promote cancer have remained an enigma. Alterations to the gut microbiota, the microorganisms that colonize the intestinal tract of humans and mice, may provide a link between obesity and PDAC, offering a previously untapped target to develop novel anti-cancer strategies in this highly lethal disease. To ascertain the importance of the obesity-associated microbiome in PDAC progression, we leveraged our previously established autochthonous model of obesity-associated PDAC (Pdx1-Cre; KrasLSL-G12D/+; LepOb/Ob) (KCO) which allows for rapid and reversible weight gain in a Kras-driven mouse tumor model that closely mimics the genetic and histologic features of human PDAC progression. Ablation of the gut microbiome via broad-spectrum antibiotics intercepted tumor development in KCO mice highlighting the importance of the gut microbiome in obesity-driven PDAC. By correlating microbial composition to tumor burden in antibiotic-treated and control mice via 16S rRNA sequencing, we are identifying microbial communities that may be causal for tumor development. In parallel studies, we performed 16S rRNA sequencing on feces serially collected from wild-type, LepOb/Ob ad libitum-fed mice (AL), and LepOb/Ob mice on calorie restriction (CR), which induces rapid weight loss. We found that obesity causes reversible changes to gut microbial composition including an increase in Proteobacteria, a phylum that holds the most known species to secrete histamine, a bioamine implicated in pancreatitis, a PDAC risk factor, and PDAC development itself. Concordant with these findings, Parallel Receptorome Expression and Screening via Transcriptional Output-SALSA (PRESTO-SALSA), a novel high-throughput system that allows for sensitive detection of microbial metabolites that exhibit mammalian GPCR agonism, feces from LepOb/Ob AL mice showed robust activation of histamine receptors (HRHs), while feces from obese mice subject to CR exhibited reduced Proteobacteria species and decreased capacity to activate mammalian HRHs, suggesting weight loss by CR mitigates microbe-derived histamine. Ongoing studies are testing whether microbe-derived histamine is necessary and sufficient for the tumor-promoting effects of obesity. Collectively, our results establish a dependency for the gut microbiome in obesity-driven PDAC, identify reversible obesity-induced microbial communities in the gut, and nominate microbial metabolites capable of activating pro-tumorigenic GPCR pathways as potential causative mechanisms. Citation Format: Christian F. Ruiz, Rylee McDonnell, Lauren Lawres, Yiyun Cao, Deguange Song, Nicole Sonnert, Noah Palm, Mandar D. Muzumdar. Obesity transforms the gut microbiome to invoke a cancer permissive state [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C048.

Abstract A100: KRAS inhibition targets and depends on pro-oncogenic HuR in PDAC cells

Abstract The Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is found in more than 90% of patients with pancreatic ductal adenocarcinomas (PDACs), making it a prime target for therapeutic drug development. HuR (ELAVL1) has been extensively studied in the context of PDAC and also plays a key role in PDAC progression and chemoresistance. In recent years, researchers have shed light on the specific role of the HuR protein in PDAC and its relationship with KRAS signaling. Using in KRASG12D – mutant patient derived models, cell lines derived from GEMM models, and conventional cell lines, we found that HuR CRISPR knock out lines showed more than 80-fold resistance to MRTX1133 (a KRAS inhibitor targeted against G12D mutation) compared to wild type cells. Furthermore, western blot and immunofluorescence studies demonstrated that therapeutic inhibition of KRAS, inhibited HuR protein expression and subcellular localization in as early as 3 hours post-treatment. Furthermore, HuR protein levels decreased post-treatment even under nutrient starvation. Herein, we show that the presence of HuR is necessary for the efficacy of KRAS targeting and Kras inhibition can rapidly degrade HuR protein levels and its active subcellular localization. Mechanistic studies determining the molecular interaction between HuR and KRAS and KRAS related proteins and mRNAs are underway. Taken together with ongoing studies, we are exploring the importance of HuR as a key downstream target of KRAS’ oncogenic activity and if the loss of HuR may be a potent, PDAC therapeutic resistance mechanism. Understanding the interplay between KRAS and HuR could offer a novel strategy to disrupt the oncogenic KRAS signaling cascade and overcome the potential inherent resistance of KRAS-mutant PDACs to promising, KRAS-targeted therapies. Citation Format: Hen Halamish, Michaela Bush, Alexander Smith, Rosalie Sears, Jonathan Brody. KRAS inhibition targets and depends on pro-oncogenic HuR in PDAC cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A100.

Abstract B063: Tertiary lymphoid organogenesis and lymphocyte activation in human organ chips

Abstract The rates of tertiary lymphoid organ (TLO) formation in pancreatic cancer are very low but patients with TLO have dramatically better survival rates. Our goal is to understand how pancreatic cancer suppresses TLO so that we can improve patient survival and reduce recurrence. We integrated human cell lines from pancreatic and lung cancer with either high or low PDL1/L2 expression into our previously described lymphoid follicle (LF) chip (Goyal et al., Adv. Sci, 2022), where peripheral blood mononuclear cells are induced to form 3D TLO-like structures. The hot lung cancer cell line, which expressed high levels of PDL1/L2 and a “hot” phenotype in published murine studies, produced a high levels of cytokines and stimulated increased TLO formation in the LF chip. In contrast, the cold lung and pancreatic cell lines did not induce the cytokine signature or TLOs. Importantly, TLO formation correlated with increased B cell activation, anti-tumor CD8 activity and tumor cell death. We have coupled this in vitro study with analysis of the pancreatic cancer transcriptome from the Cancer Genome Atlas to identify therapeutic targets to promote TLO formation in pancreatic cancer. In ongoing studies, we are assessing the functionality of the lung cancer TLO in vitro and in vivo and identifying therapeutics that may improve TLO formation in pancreatic cancer. Citation Format: Yunhao Zhai, Pranav Prabhala, Lucas Barck, Min Wen Ku, Aditya Patil, Viktor Horvath, Russell A. Gould, Donald E. Ingber, Girija Goyal. Tertiary lymphoid organogenesis and lymphocyte activation in human organ chips [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B063.

Abstract A073: A link between the mitochondrial enzyme glutamic-oxaloacetic transaminase 2 (GOT2) and epigenetic dysregulation in pancreatic cancer

Abstract At present, pancreatic cancer stands as the 3rd cause of cancer-related fatalities in the US. The most common type of pancreatic cancer develops from the exocrine pancreas known as ductal adenocarcinoma (PDAC). PDAC exhibits a highly aggressive nature and carries a bleak prognosis, with a 5-year survival rate averaging below 10%. Moreover, the majority of patients receive their diagnosis at advanced stages of the disease. Compounding the issue, the emergence of resistance to modern first-line chemotherapies contributes to the progression of PDAC and exacerbates its overall high mortality rate. The specific cause of PDAC remains unknown, although lifestyle factors such as alcohol and tobacco use, along with genetic abnormalities, have been linked to its increasing incidence over the past decade. Mutations in KRAS, TP53, SMAD4, and CDKN2A are recognized as genetic drivers contributing to the progression of PDAC. Recently, research has shifted its focus to investigating the role of epigenetic dysregulation in PDAC advancement. Significant evidence suggests that remodeling of the epigenome plays a crucial role in the development and progression of PDAC, but the exact mediators responsible for these alterations remain largely unknown. Recent studies have indicated that mitochondrial enzymes may influence epigenetic modifications, thus contributing to the progression of tumorigenesis. In our previous work, we reported that the mitochondrial protein GOT2 (glutamic-oxaloacetic transaminase) supports PDAC development by promoting an immunosuppressive phenotype within the tumor microenvironment. Ongoing epigenetic studies following ChIP- and ATAC-seq (chromatin immunoprecipitation and assay for transposase-accessible chromatin with sequencing, respectively) experiments in mammalian PDAC cell lines have revealed that loss of GOT2 leads to genome-wide changes in histone acetylation and chromatin accessibility. These changes in acetylation and chromatin architecture imply a reconfiguration of the epigenetic landscape and subsequent alterations in the PDAC transcriptome, suggesting a potential role for GOT2 in maintaining epigenetic profiles within the context of pancreatic cancer. Further exploration of the genomic regions where these histone modification changes occur has implicated cis-regulatory regions and the expression of their target genes, which may be crucial for PDAC progression. Collectively, these epigenetic studies hold the potential to identify novel therapeutic targets that could address the urgent need for more effective antineoplastic treatments against this aggressive form of pancreatic cancer. Citation Format: Luis Francisco Diaz, Mara H. Sherman, Jonathan R. Brody. A link between the mitochondrial enzyme glutamic-oxaloacetic transaminase 2 (GOT2) and epigenetic dysregulation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A073.

Abstract B096: Acquired resistance to KRAS inhibition modulates the pancreatic cancer tumor microenvironment

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with dismal patient outcomes. Acquired resistance to chemotherapeutics is a major barrier to successful pancreatic cancer treatment. MRTX-1133 is a potent, selective KRAS G12D inhibitor that is in early-phase clinical trials. While initially effective, clinical experience with KRAS G12C inhibitors has shown tumors rapidly acquire therapeutic resistance. The tumor microenvironment (TME) plays a pivotal role in resistance development by activating prosurvival pathways, promoting epithelial-mesenchymal transition (EMT) and stemness, and inhibiting apoptosis. In this study, we sought to understand changes in the PDAC TME associated with acquired resistance to KRAS G12D inhibition. Methods: In order to investigate resistance to KRAS G12D inhibition in PDAC, we developed various mouse and human MRTX-1133 resistant (MRTXR) cell models (FC1199, FC1242, FC1245, and SUIT2). Intriguingly, these models also displayed resistance to several pan-RAS(ON) inhibitors. We utilized flow cytometry analysis to investigate the dynamic changes occurring in the TME following resistance to MRTX-1133, and we employed cytokine array and RNA-seq analyses to elucidate the impact of cancer cells on gene and protein expression of TME cells. Results: A comparative analysis between parental PDAC cells and MRTXR tumors revealed significant alterations within the TME. We found a significant decrease (p=0.013, p=0.045, respectively) in the number of inactivated and naïve CD8 T cells (CD8+/CD69+ and CD44-/CD62L+ cells) in MRTXR tumors. We also observed an upregulation of CXCL1, CCL20, and CXCL2 secretion in MRTXR cells compared to parental cells. Conclusions: Pancreatic cancer cells with acquired resistance to KRAS inhibitors modulate their tumor microenvironment uniquely. Ongoing studies aim to identify the critical pathways responsible for these alterations and to develop alternative treatment strategies for KRAS therapy-resistant tumors. Citation Format: Kevin Christian Montecillo Gulay, Jay Patel, Isabella Ng, Tatiana Hurtado de Mendoza, Herve Tiriac, Andrew Lowy. Acquired resistance to KRAS inhibition modulates the pancreatic cancer tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B096.

A modern approach to the study of the problem of pathogenesis and prevention venous thromboembolic complications in patients with trauma (literature review)

Relevance. Patients with trauma are at increased risk of venous thromboembolic complications (VTE), which includes deep vein thrombosis of the lower extremities (DVT) and pulmonary embolism (PE). Within surgical and trauma clinics, thromboembolic complications are the most common preventable cause of in-hospital mortality. Numerous studies have shown that the incidence rate of deep vein thrombosis (DVT) in trauma patients without prevention varies from 5 to 80%.Aim. To analyze the pathogenesis of the development of venous thrombosis and explore existing approaches to prevent VTE in trauma patients.Materials and methods. In the course of the work, topical literary sources of domestic and foreign authors on a given topic were analyzed.Results. The mechanism of thrombus formation in trauma involves slowing blood flow, damage to the vessel wall and impaired blood clotting in the form of hypercoagulation. The state of hypercoagulation combined with prolonged immobility increases the risk of VTE. LMWH is the treatment of choice for the prevention of VTE in trauma. Today, there are ongoing studies related to the correction of preventive doses of LMWH and the search for a single standard, depending on numerous factors.Conclusion. Severity and multiplicity of lesions correlate with severity of systemic inflammation, hypercoagulation, and incidence of venous thrombosis. However, this correlation has not been sufficiently studied in the scientific literature. The search for specific methods for the prevention of deep vein thrombosis leading to PE is an urgent problem of surgery of injuries both peaceful and wartime.

Effect of Oxygen Tension on Glioblastoma Cell Growth

Background and Hypothesis:Glioblastoma (GBM) is an aggressive primary malignancy of the CNS with a dismal prognosis (~15-20 months) despite standard of care therapies. The poor prognosis of GBM despite ongoing research may be due to inaccuracies in preclinical models of the disease. Cell lines typically used to study GBM are exposed to ambient air containing 21% oxygen (normoxia compared to physiologic oxygen tension, physoxia, ~5%). In extracranial tumors, exposure to the oxygen in ambient air triggers epigenetic changes that alter cell growth, metabolism, and treatment responsiveness. The aim of this study is to obtain preliminary data on the impact ofphysiologic oxygen tensions on primary glioma cell growth in vitro.
 Experimental Design:Growth of primary glioma cell lines (GB43, GB10, GB001) and one immortalized cell line (293T HEK) was assessed in normoxia and physoxia. All cell lines were plated at 10,000 cells per well. Cells were harvested and counted in triplicate on days 2, 4, 6, 8, 10, 12. On each day the cells were counted, the media in the remaining wells was changed. This experiment was repeated three times. Wound healing assays with all cell lines were also performed at normoxia and physoxia.
 Results:Cells line growth curves were plotted and showed consistent exponential growth after counting on day two. From these graphs, the cell doubling time was calculated over a period of four days during which the cells were undergoing exponential growth.
 Conclusions:Current results indicated that primary glioblastoma cell lines grow at different rates at differing oxygen tensions. In ongoing studies, we are exploring the effect of low oxygen tensions on functional assays such as wound-healing. Future work will assess rates of growth and functional consequence of physoxia in tumor samples never exposed to ambient air to most accurately recapitulate the in-situ environment.

Common design and data elements reported in active mechanical thrombectomy trials focusing on distal medium vessel occlusions and minor strokes: a systematic review

BackgroundDistal medium vessel occlusions (DMVOs) and minor strokes represent emerging frontiers in mechanical thrombectomy (MT). Although several randomized clinical trials (RCTs) are underway, the design characteristics of these trials and...

Longitudinal characterization of the captive adult and tadpole Wyoming toad (Anaxyrus baxteri) microbiome

IntroductionAt one time thought to be extinct in the wild, the Wyoming toad (Anaxyrus baxteri) is one of the most critically endangered North American amphibian species. Despite approximately 20 years of ex situ breeding and reintroduction programs, this species remains functionally extinct in the wild. There is concern among those working in these programs that individuals bred in captivity fail to develop a microbiome that withstands the stressors of their native habitat following release. In related species, the skin microbiome has been shown to have a defensive function against common pathogens affecting these animals. However, the early-life microbiome of developing tadpoles in this species remains unknown and, therefore, this defensive function is unexplored in the Wyoming toad.MethodsThis study employed 16S rRNA amplicon sequencing to document the baseline microbiome of tadpole and adult Wyoming toads bred for release at Omaha’s Henry Doorly Zoo and Aquarium (Omaha, Nebraska, USA). We characterized the development of the mucosal microbiome in tadpoles and the mucosal and cloacal microbiome of adult toads.ResultsOur results revealed significant differences between tadpole and adult microbiomes, as well as significant sex-dependent differences within the adult Wyoming toads, in terms of richness and composition.DiscussionThese findings have identified the baseline microbiome of this endangered species and variables significantly influencing its composition in captivity. Ongoing studies of the only extant wild population are expected to identify taxa not found in captive toads, and potentially help 100 design husbandry modifications to maximize survivability following reintroduction to the wild.

Bispecific antibodies in indolent B-cell lymphomas.

The advent of immunotherapy in lymphomas, beginning with Rituximab, have led to paradigm shifting treatments that are increasingly bringing a greater number of affected patients within the ambit of durable disease control and cure. Bispecific antibodies harness the properties of the immunoglobulin antibody structure to design molecules which, apart from engaging with the target tumour associated antigen, engage the host's T-cells to cause tumour cell death. Mosunetuzumab, an anti-CD20 directed bispecific antibody was the first to be approved in follicular lymphoma, this has now been followed by quick approvals of Glofitamab and Epcoritamab in diffuse large B-cell lymphomas. This article reviews contemporary data and ongoing studies evaluating the role of bispecific antibodies in indolent b-cell non Hodgkin lymphomas. This is an area of active research and presents many opportunities in advancing the treatment of indolent lymphomas and potentially forge a chemo-free treatment paradigm in this condition.

Unlocking the potential of dCas9: Epigenetic targeting in cancer immunotherapy

Cancer's profound medical challenge underscores the urgency for innovative solutions. Contemporary immunotherapy has formidable challenges dealing with immune evasion, complex tumor microenvironments (TME), and checkpoint inhibitors. Improvements to immunotherapy have been made through genome editing for which the Cas9 system is utilized, but its reliance on DNA repair mechanisms coupled with elevated off-target effects presents limitations. One alternative approach to gene editing is the modulation of gene expression using dCas9 and associated effectors. An advantage is dCas9's non-cleaving nature which reduces the chance of off-target effects and avoids permanent changes to the genome, offering enhanced safety as a gene modulation tool. We reviewed ongoing studies and clinical trials on immunotherapy, gene expression, and dCas9 to address whether gene modulation via dCas9 could be a viable solution to improve gene therapy. In (one/a few) studies found that dCas9's adoption decreases off-target effects, heightening its appeal for improving immunotherapy compared to traditional gene editing with Cas9. Therefore, integrating dCas9 in suppressing key target genes, dcas9 with effector domains or epigenetic modifiers could prove as a pivotal strategy, enabling precise gene regulation and epigenetic modification. Specifically, transcriptional activators like VP64 and repressors like KRAB exhibit the potential to modulate target genes when used with dcas9. Acknowledging limitations, such as mutations affecting gene regulation, this study underscores the transformative potential of dCas9-mediated epigenetic regulation in reshaping the landscape of cancer treatment and calls for further investigation to unlock its full therapeutic potential.

Targeted Therapies in Cancer Treatment: Unveiling the Latest Breakthroughs and Promising Approaches

This review article delves into the realm of cancer treatment, specifically focusing on targeted therapies. It aims to present the most recent breakthroughs and promising approaches in this rapidly evolving field. Targeted therapies have emerged as a revolutionary approach in cancer treatment, aiming to selectively and precisely attack cancer cells while sparing normal tissues. This article explores various targeted therapy strategies, including monoclonal antibodies, small molecule inhibitors, immunotherapies, and gene therapies. In recent years, there have been significant advancements in understanding the molecular and genetic basis of cancer, which has led to the identification of novel therapeutic targets. The article sheds light on these newly discovered targets and highlights their potential in designing more effective and personalized treatment regimens for cancer patients. Furthermore, the review addresses the challenges and limitations associated with targeted therapies, such as resistance mechanisms and the heterogeneity of tumors. Strategies to overcome these obstacles are discussed, including combination therapies and the development of next-generation targeted agents. The role of precision medicine in cancer treatment is also explored, emphasizing the importance of biomarker-guided therapy selection to optimize treatment outcomes. Additionally, the review touches upon the integration of targeted therapies with conventional treatments, such as chemotherapy and radiation therapy, to enhance overall treatment efficacy. Finally, the article examines ongoing clinical trials and preclinical studies that are investigating cutting-edge targeted therapies, showcasing the potential impact of these approaches in transforming cancer care. In conclusion, targeted therapies in cancer treatment represent a rapidly expanding field with remarkable breakthroughs and promising avenues. Understanding the latest advancements and challenges in this domain is essential to harness the full potential of targeted therapies and ultimately improve patient outcomes in the battle against cancer.

Abstract B006: Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors?

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest malignancy of the pancreas, with an overall five-year survival rate of only 12%. Despite therapeutic advances, effective therapies are still limited. It is now known that many of the DNA damage repair (DDR) genes that are mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway. Loss-of-function alterations in DDR genes such as BRCA1/2 mutations render PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi) and platinum-based agents. Since this treatment strategy is currently limited to only 5-10% of PDAC patients, it underscores a dire need to find new therapeutic avenues and expand the PDAC patient population that could benefit from these therapies. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1 in the process of HRR, is upregulated in PDAC cells exposed to PARPi/platinums. PDAC tissue microarray stained for BARD1 also shows higher BARD1 expression in PDAC tissues compared to normal pancreas. Here, we explored the effects of targeting BARD1 on PDAC cells. For this, we created genetic models of BARD1 inhibition and utilized these model systems to study if loss of BARD1 slows PDAC growth, invasion, and chemo-sensitizes PDAC cells to PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. Loss of BARD1 protein and mRNA expression was confirmed by western blot and qRT-PCR analyses. Using colony formation assays, Pico green cell survival assays, and Boyden matrigel invasion assays, we found that inhibition of BARD1 resulted in slower growth and decrease in invasion of pancreatic cancer cells in vitro. The expression levels of EMT markers such as zeb-1 and vimentin, were also decreased. These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 CRISPR KO (+/- and -/-) and WT PDAC cells were injected subcutaneously on flanks of mice and tumor growth was monitored over time. Kaplan Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (P=0.026) delayed PDAC tumor growth in vivo and extended survival (P<0.001). CRISPR KO of BARD1 also enhanced the efficacies of PARP inhibitors like olaparib, rucaparib and veliparib, and lowered their IC50s (fold changes >10). Our work indicates that targeting BARD1 affects key hallmarks of cancer (growth and invasion), such that the window of therapeutic opportunity is extended, which further allows chemo-sensitization of PDAC cells to DNA damaging agents. Our ongoing studies will explore if BARD1 loss is a predictive marker of PDAC sensitivity to other DNA-damaging agents besides PARPi and allow the development of therapeutic strategies to target BARD1 in PDAC. Citation Format: Aditi Jain, Rutuj P. Kusurkar, Eleanor Jenkins, Avinoam Nevler, Wei Jiang, Charles J. Yeo. Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B006.

Publication of Results of Registered Trials With Published Study Protocols, 2011-2022

Publishing study protocols might reduce research waste because of unclear methods or incomplete reporting; on the other hand, there might be few additional benefits of publishing protocols for registered trials that are never completed or published. No study has investigated the proportion of published protocols associated with published results. To estimate the proportion of published trial protocols for which there are not associated published results. This cross-sectional study used stratified random sampling to identify registered clinical trials with protocols published between January 2011 and August 2022 and indexed in PubMed Central. Ongoing studies and those within 1 year of the primary completion date on ClinicalTrials.gov were excluded. Published results were sought from August 2022 to March 2023 by searching ClinicalTrials.gov, emailing authors, and using an automated tool, as well as through incidental discovery. The primary outcome was a weighted estimate of the proportion of registered trials with published protocols that also had published main results. The proportion of trials with unpublished results was estimated using a weighted mean. From 1500 citations that were screened, 308 clinical trial protocols were included, and it was found that 87 trials had not published their main results. Most included trials were investigator-initiated evaluations of nonregulated products. When published, results appeared a mean (SD) of 3.4 (2.0) years after protocol publications. With the use of a weighted mean, an estimated 4754 (95% CI, 4296-5226) eligible clinical trial protocols were published and indexed in PubMed Central between 2011 and 2022. In the weighted analysis, 1708 of those protocols (36%; 95% CI, 31%-41%) were not associated with publication of main results. In a sensitivity analysis excluding protocols published after 2019, an estimated 25% (95% CI, 20%-30%) of 3670 (95% CI, 3310-4032) protocol publications were not associated with publication of main results. This cross-sectional study of clinical trial protocols published on PubMed Central between 2011 and 2022 suggests that many protocols were not associated with subsequent publication of results. The overall benefits of publishing study protocols might outweigh the research waste caused by unnecessary protocol publications.

Comparative Effectiveness of Neoadjuvant Pembrolizumab Versus Cisplatin-based Chemotherapy or Upfront Radical Cystectomy in Patients with Muscle-invasive Urothelial Bladder Cancer

BackgroundRecent progresses in the use of immune checkpoint inhibitor (ICI) have challenged the therapeutic standards in patients with muscle-invasive urothelial bladder carcinoma (MIBC). ObjectiveTo compare neoadjuvant pembrolizumab followed by radical cystectomy (RC) versus neoadjuvant chemotherapy (NAC) and RC or upfront RC, according to cisplatin eligibility. Design, setting, and participantsWe conducted two separate analyses for cisplatin-eligible and cisplatin-ineligible cT2-4N0M0 MIBC patients. We used a propensity score adjustment that relied on inverse probability of treatment-weighting (IPTW). InterventionPembrolizumab within the PURE-01 trial, and NAC and RC or upfront RC from a high-volume tertiary care referral center. Outcome measurements and statistical analysisThe primary endpoint in both analyses was event-free survival (EFS), defined as freedom from recurrence, and/or death from any cause indexed from the date of treatment initiation or RC. The secondary endpoints included EFS in propensity score–matched patients, pathologic response rate, and recurrence-free survival (RFS) after RC. Results and limitationsA total of 458 patients who underwent RC, with or without NAC, at Moffitt Cancer Center between October 2005 and October 2020, and 146 patients enrolled in PURE-01 were analyzed. In cisplatin-ineligible patients, EFS was superior in those receiving pembrolizumab (p < 0.001). The estimated 3-yr EFS was 77.8% (95% confidence interval [CI]: 63.5–95.2) for pembrolizumab and RC, and 36.1% (95% CI: 28.6–45.5) for upfront RC. EFS remained superior in those receiving neoadjuvant ICI (NICI) following IPTW (p < 0.001). In cisplatin-eligible patients, EFS was superior in those receiving pembrolizumab and RC (p < 0.001). The estimated 3-yr EFS was 86.9% (95% CI: 80.9–93.3) for pembrolizumab and 63.5% (95% CI: 56.5–71.4) for NAC. EFS remained superior in those receiving NICI following IPTW (p < 0.001). Pathologic responses and RFS in pembrolizumab-treated patients were also superior to those in NAC-treated patients. Results are limited by the retrospective nature of the study. ConclusionsIn the first ever reported comprehensive comparison of outcomes between neoadjuvant ICI and NAC, followed by RC, or upfront RC, we report increased responses and improved oncologic outcomes with neoadjuvant ICI in patients with MIBC. Patient summaryWe compared the results obtained from the use of pembrolizumab and radical cystectomy with standard-of-care treatments in patients with bladder carcinoma infiltrating the muscle layer. We reported increased response and survival rates possibilities with the use of immunotherapy, anticipating the possibility to set new therapeutic standards in these patients, pending the results of ongoing randomized studies.

The Population-Attributable Fractions of Small-for-Gestational-Age Births: Results from the Japan Birth Cohort Consortium.

A fetal growth restriction is related to adverse child outcomes. We investigated risk ratios and population-attributable fractions (PAF) of small-for-gestational-age (SGA) infants in the Japanese population. Among 28,838 infants from five ongoing prospective birth cohort studies under the Japan Birth Cohort Consortium, two-stage individual-participant data meta-analyses were conducted to calculate risk ratios and PAFs for SGA in advanced maternal age, pre-pregnancy underweight, and smoking and alcohol consumption during pregnancy. Risk ratio was calculated using modified Poisson analyses with robust variance and PAF was calculated in each cohort, following common analyses protocols. Then, results from each cohort study were combined by meta-analyses using random-effects models to obtain the overall estimate for the Japanese population. In this meta-analysis, an increased risk (risk ratio, [95% confidence interval of SGA]) was significantly associated with pre-pregnancy underweight (1.72 [1.42-2.09]), gestational weight gain (1.95 [1.61-2.38]), and continued smoking during pregnancy (1.59 [1.01-2.50]). PAF of underweight, inadequate gestational weight gain, and continued smoking during pregnancy was 10.0% [4.6-15.1%], 31.4% [22.1-39.6%], and 3.2% [-4.8-10.5%], respectively. In conclusion, maternal weight status was a major contributor to SGA births in Japan. Improving maternal weight status should be prioritized to prevent fetal growth restriction.

Identification of Dynamic Force Coefficients for an Additively Manufactured Hermetic Squeeze Film Bearing Support Damper Utilizing a Pass-Through Channel

Abstract The following paper presents breakthrough experimental results for a new hermetic squeeze film damper (HSFD) concept that is integrally designed within an externally pressurized tilting-pad radial gas bearing support. The flexibly damped gas bearing module was designed for a 7.2″ (183 mm) diameter shaft and fabricated using direct metal laser melting (DMLM); also known as additive manufacturing. The bearing and HSFD were sized based on ongoing studies for oil-free supercritical carbon dioxide (sCO2) power turbines in the 8.5 MW–10 MW power range. The development of the new damper concept was motivated by past dynamic testing on HSFD, which generated frequency-dependent stiffness and damping force coefficients. In efforts to eliminate the frequency dependency, a new HSFD architecture was conceived that adds accumulator volumes and a pass-through channel to previously conceived HSFD flow network designs. The other motivation for the work is the need to develop a cost-effective and reliable oil-free bearing technology that is scalable to large power turbomachinery applications. There were several objectives for the following work. The first objective was to successfully design and fabricate a single piece bearing-damper using additive manufacturing, while dimensionally controlling critical design features. The paper discusses the manufacturing steps and shows cut-ups that reveal adequate clearance control capability with internal damper clearances. The second objective was to perform experimental testing with the new HSFD design in efforts to extract stiffness and damping coefficients for excitation frequencies within 20–160 Hz and peak vibration amplitudes between 0.25 mils (6.35 microns) to 1 mil (25.4 microns). The test results for a single HSFD bearing module indicated that the design modifications to the HSFD architecture were successful in eliminating nearly all the frequency dependencies for the stiffness force coefficient. The dynamic tests yielded a stiffness coefficient that varied between 112 klb/in. (19.6 MN/m) and 96 klb/in. (16.8 MN/m). The damping force coefficient however, exhibited relatively more variation with frequency with values residing between 175 lb-s/in. (31 kN-s/m) to 214 lb-s/in. (37 kN-s/m). Finally, the paper advances a three-dimensional fluid-structure interaction (FSI) model using transient finite element analysis (FEA) coupled to a computational fluid dynamics (CFD) model. The FSI analysis performed between 20 Hz and 80 Hz was used to predict the stiffness and damping of the HSFD using a quarter-section model of the damper. The FSI analysis was able to support test results by showing only a 6–7.4% change in the magnitude of force coefficients. Stiffness predictions agree reasonably well with experiments whereas damping is underpredicted.