Target In Oncology Research Articles

Undifferentiated Sarcomatoid Carcinoma of the Pancreas: From Histology and Molecular Pathology to Precision Oncology.

Undifferentiated sarcomatoid carcinoma of the pancreas (SCP) is a rare and aggressive subtype of pancreatic cancer. Histologically, SCP is a poorly differentiated tumor characterized by the lack of glandular differentiation and the presence of mesenchymal-like, spindle-shaped tumor cells. Due to its rarity, only sporadic cases have been reported, while its molecular characterization has not been sufficiently described. Surgical resection with curative intent is the gold-standard of SCP management, but this strategy is possible only in a small proportion of cases due to SCP early metastasization. Although SCP is generally associated with a poor prognosis, some clinical cases amenable to surgical resection and followed by adjuvant chemotherapy have demonstrated a remarkably long survival. Preliminary molecular insights on the SCP molecular landscape have demonstrated the recurrent presence of KRAS and TP53 mutations, highlighting genetic similarities with conventional pancreatic ductal adenocarcinoma (PDAC). Although the use of immunotherapy in PDAC remains an unmet challenge, recent insights indicated a potentially significant role of the PD-L1/Notch3 axis in SCP, opening new horizons for immunotherapy in this cancer subtype. In this review, we described the most important clinic-pathologic features of SCP, with a specific focus on their molecular landscape and the potential targets for precision oncology.

DNA damage response proteins in canine cancer as potential research targets in comparative oncology

The DNA damage response (DDR) is a complex signal transduction network that is activated when endogenous or exogenous genotoxins damage or interfere with the replication of genomic DNA. Under such conditions, the DDR promotes DNA repair and ensures accurate replication and division of the genome. High levels of genomic instability are frequently observed in cancers and can stem from germline loss‐of‐function mutations in certain DDR genes, such as BRCA1, BRCA2, and p53, that form the basis of human cancer predisposition syndromes. In addition, mutation and/or aberrant expression of multiple DDR genes are frequently observed in sporadic human cancers. As a result, the DDR is considered to represent a viable target for cancer therapy in humans and a variety of strategies are under investigation. Cancer is also a significant cause of mortality in dogs, a species that offers certain advantages for experimental oncology. Domestic dogs present numerous inbred lines, many of which display predisposition to specific forms of cancer and the study of which may provide insight into the biological basis of this susceptibility. In addition, clinical trials are possible in dogs and may lead to therapeutic insights that could ultimately be extended to humans. Here we review what is known specifically about the DDR in dogs and discuss how this knowledge could be extended and exploited to advance experimental oncology in this species.

Circulating Tumor Cell Transcriptomics as Biopsy Surrogates in Metastatic Breast Cancer

BackgroundMetastatic breast cancer (MBC) and the circulating tumor cells (CTCs) leading to macrometastases are inherently different than primary breast cancer. We evaluated whether whole transcriptome RNA-Seq of CTCs isolated via an epitope-independent approach may serve as a surrogate for biopsies of macrometastases.MethodsWe performed RNA-Seq on fresh metastatic tumor biopsies, CTCs, and peripheral blood (PB) from 19 newly diagnosed MBC patients. CTCs were harvested using the ANGLE Parsortix microfluidics system to isolate cells based on size and deformability, independent of a priori knowledge of cell surface marker expression.ResultsGene expression separated CTCs, metastatic biopsies, and PB into distinct groups despite heterogeneity between patients and sample types. CTCs showed higher expression of immune oncology targets compared with corresponding metastases and PB. Predictive biomarker (n = 64) expression was highly concordant for CTCs and metastases. Repeat observation data post-treatment demonstrated changes in the activation of different biological pathways. Somatic single nucleotide variant analysis showed increasing mutational complexity over time.ConclusionWe demonstrate that RNA-Seq of CTCs could serve as a surrogate biomarker for breast cancer macrometastasis and yield clinically relevant insights into disease biology and clinically actionable targets.

USP14: Structure, Function, and Target Inhibition.

Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), is associated with proteasomes and exerts a dual function in regulating protein degradation. USP14 protects protein substrates from degradation by removing ubiquitin chains from proteasome-bound substrates, whereas promotes protein degradation by activating the proteasome. Increasing evidence have shown that USP14 is involved in several canonical signaling pathways, correlating with cancer, neurodegenerative diseases, autophagy, immune responses, and viral infections. The activity of USP14 is tightly regulated to ensure its function in various cellular processes. Structural studies have demonstrated that free USP14 exists in an autoinhibited state with two surface loops, BL1 and BL2, partially hovering above and blocking the active site cleft binding to the C-terminus of ubiquitin. Hence, both proteasome-bound and phosphorylated forms of USP14 require the induction of conformational changes in the BL2 loop to activate its deubiquitinating function. Due to its intriguing roles in the stabilization of disease-causing proteins and oncology targets, USP14 has garnered widespread interest as a therapeutic target. In recent years, significant progress has been made on identifying inhibitors targeting USP14, despite the complexity and challenges in improving their selectivity and affinity for USP14. In particular, the crystal structures of USP14 complexed with IU1-series inhibitors revealed the underlying allosteric regulatory mechanism and enabled the further design of potent inhibitors. In this review, we summarize the current knowledge regarding the structure, regulation, pathophysiological function, and selective inhibition of USP14, including disease associations and inhibitor development.

Clinical Translation: Targeting the Estrogen Receptor.

Estrogen Receptor alpha (ERα) stands as one of the most successfully prosecuted drug targets in oncology, beginning with the approval of tamoxifen for women with ERα positive (ER+) breast cancer over 40years ago. The field continued to advance with the development of aromatase inhibitors and the pure antiestrogen fulvestrant. With multiple endocrine therapies approved for the treatment of ER+ breast cancer, efforts to generate novel ERα-targeted therapeutics somewhat diminished in the early 2000s. Today however, there are at least eight new molecular entities targeting ERα under active clinical investigation, each with the aim of bringing further benefit to patients. This remarkable re-energizing of the field was spurred in part by the discovery of highly prevalent ERα mutations as a mechanism of resistance to standard-of-care therapies, which provided unequivocal evidence of the continued, and broad, dependence of tumors on ERα, despite relapsing after earlier lines of endocrine therapy. Re-engagement of the pharmaceutical and biotechnology industries with ERα as a drug target has been further underpinned by the impressive advances made in medicinal chemistry, enabling desirable mechanistic features - high potency full ERα antagonism - to be combined with improved drug-like properties - oral bioavailability and optimized pharmacokinetics. In this chapter, we describe the rich history and science behind the currently evolving landscape of ERα targeting in breast cancer.

Systematic Pan-Cancer Characterization of Nuclear Receptors Identifies Potential Cancer Biomarkers and Therapeutic Targets

The nuclear receptor (NR) superfamily is one of the major druggable gene families, representing targets of approximately 13.5% of approved drugs. Certain NRs, such as estrogen receptor and androgen receptor, have been well demonstrated to be functionally involved in cancer and serve as informative biomarkers and therapeutic targets in oncology. However, the spectrum of NR dysregulation across cancers remains to be comprehensively characterized. Through computational integration of genetic, genomic, and pharmacologic profiles, we characterized the expression, recurrent genomic alterations, and cancer dependency of NRs at a large scale across primary tumor specimens and cancer cell lines. Expression levels of NRs were highly cancer-type specific and globally downregulated in tumors compared with corresponding normal tissue. Although the majority of NRs showed copy-number losses in cancer, both recurrent focal gains and losses were identified in select NRs. Recurrent mutations and transcript fusions of NRs were observed in a small portion of cancers, serving as actionable genomic alterations. Analysis of large-scale CRISPR and RNAi screening datasets identified 10 NRs as strongly selective essential genes for cancer cell growth. In a subpopulation of tumor cells, growth dependencies correlated significantly with expression or genomic alterations. Overall, our comprehensive characterization of NRs across cancers may facilitate the identification and prioritization of potential biomarkers and therapeutic targets, as well as the selection of patients for precision cancer treatment. SIGNIFICANCE: Computational analysis of nuclear receptors across multiple cancer types provides a series of biomarkers and therapeutic targets within this protein family.

Dual-specificity phosphatases: therapeutic targets in cancer therapy resistance.

Therapy resistance is the principal obstacle to achieving cures in cancer patients and its successful tackling requires a deep understanding of the resistance mediators. Increasing evidence indicates that tumor phosphatases are novel and druggable targets in translational oncology and their modulation may hinder tumor growth and motility and potentiate therapeutic sensitivity in various neoplasms via regulation of various signal transduction pathways. Dual-specificity phosphatases (DUSPs) are key players of cell growth, survival and death and have essential roles in tumor initiation, malignant progression and therapy resistance through regulation of the MAPK signaling pathway. In this review, different aspects of DUSPs are discussed. A comprehensive literature review was performed using various websites including PubMed. We provide mechanistic insights into the roles of well-known DUSPs in resistance to a wide range of cancer therapeutic approaches including chemotherapy, radiation and molecular targeted therapy in human malignancies. Moreover, we discuss the development of DUSP modulators, with a focus on DUSP1 and 6 inhibitors. Ultimately, the preclinical investigations of small molecule inhibitors of DUSP1 and 6 are outlined. Emerging evidence indicates that the DUSP family is aberrantly expressed in human malignancies and plays critical roles in determining sensitivity to a wide range of cancer therapeutic strategies through regulation of the MAPK signaling pathways. Consequently, targeting DUSPs and their downstream molecules can pave the way for more effective cancer therapies.

Abstract PO-137: Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race

Abstract African American (AA) men exhibit 2-3 times higher mortality from prostate cancer compared with Caucasian American (CA) men. Factors contributing to the disparity include societal-, neighborhood- and institutional-level determinants of health. In addition, a number of studies have reported individual-level ancestry-related biological differences, including in mutations, copy number variation, aggregate gene expression and response to treatment between AA and CA prostate cancer patients. Previously, by comparing the transcriptome between 20 AA and 15 CA prostate cancer patients, we identified a large number of race-related Alternative RNA Splicing (ARS) events. Among these, we further demonstrated that an exon skipping event involving exon 20 within PIK3CD increased tumor growth rate, metastatic potential and drug resistance in prostate cancer. To expand our previous findings, we collected non-neoplastic and tumor-paired tissue from 37 AA and 40 CA prostate cancer patients with different Gleason score categories: 14 high grade (4 AA and 10 CA), 22 low grade (10 AA and 12 CA), and 41 intermediate grade (23 AA and 18 CA). DNA and RNA were isolated for ancestral genotyping and RNA seq analysis, respectively. To achieve RNA sequencing depth adequate for ARS analysis, we performed RNA seq of 150 bp paired-end and an average of 5 × 106 reads per sample. The read alignment was done using Star 2 TwoPass pipeline. The rMATS pipeline was used for ARS annotation and quantification. We identified 105,403 ARS events, including 60,657 exon skipping, 17,439 alternative acceptor, 12,737 alternative donor, 9,555 retained intron and 5,015 mutually exclusive exon. Using the Wilcoxon rank-sum test, we compared the Percent Spliced In (PSI) between AA and CA of the same Gleason score category and identified ARS events exhibiting ΔPSI > 15% and p-value < 0.05. Specifically, we identified 536 race-related ARS events in high grade, 492 race-related ARS events in low grade, and 447 race-related ARS events in intermediate grade. Gene Ontology analysis demonstrated that the genes undergoing race-related ARS events function in cellular processes relevant to cancer biology, including metabolic processes in low grade, NF-kappaB signaling in intermediate grade and cell motility in high grade. Specific examples of these genes include ERG and PARP2 in high grade, KLK2 and DNMT1 in intermediate grade, and AURKA and SEMA3A in low grade. These findings support a potential role for the ARS process in diversifying gene expression, potentially contributing to prostate cancer aggressiveness in AA patients. The race-related ARS events identified in our work represent potential biomarkers and/or therapeutic targets for precision oncology in the context of prostate cancer. Further analysis of the function of prioritized race-related ARS events and their association with local ancestry is ongoing. Funding: DoD Prostate Cancer Research Program Health Disparity Research Award. NIH Basic Research in Cancer Health Disparities R01 Award. Prostate Cancer Foundation Movember Challenge Award. Citation Format: Muthana Al Abo, Wen-Chi Foo, Daniel J. George, Steven R. Patierno, Jennifer A. Freedman. Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-137.

Preliminary assessment of the possibility of using urokinase uPA and urokinase receptor uPAR as universal diagnostic criteria in patients with colorectal and gastric adenocarcinoma

Background. Lack of universal diagnostic parameters that can accurately and reliably diagnose presence of a malignant neoplasm, anticancer drug effectiveness or metastasis development including dormant ones and also to detect the progression or relapse of the disease at an early stage put on the first place studies related to the identification of such markers. Increased secretion and activity of the urokinase type plasminogen activator (uPA), its receptor (uPAR) accompany many types of malignant neoplasms, contributing their progression and metastasis, as well as the emergence of chemoresistance. So today these proteins are promising diagnostic targets in oncology.
 Aim. To evaluate the diagnostic significance of the expression levels of uPA and uPAR in blood serum of patients with colorectal and gastric adenocarcinoma and uPA/uPAR distribution in samples obtained from the primary tumor node, to assess the possibility of their use as universal diagnostic indicators in cancer patients at the stage of primary treatment.
 Materials and methods. The study included patients with colorectal and gastric adenocarcinoma in the amount of 50 people and 25 healthy volunteers. The content of uPA and uPAR in blood serum was assessed by enzyme-linked immunosorbent assay, the level of expression of the studied proteins in tumor tissue was assessed using immunohistochemical staining with antibodies to uPA and uPAR.
 Results. It was found that in comparison with healthy donors uPA serum concentration was significantly higher in all patients with adenocarcinoma, regardless of gender, and the uPAR content in the blood serum was significantly higher in women. Immunohistochemical staining data showed that the expression of these two proteins in tumor tissue significantly exceeds their expression in normal tissue.
 Conclusion. Based on the obtained data the possibility of using uPA in blood serum as a universal diagnostic criterion in patients with colorectal and gastric adenocarcinoma regardless of gender was shown and uPAR in female patients.

Design, synthesis, and biological evaluation of coumarin analogs as novel LSD1 inhibitors.

The abnormal expression of lysine-specific histone demethylase 1 (LSD1) is associated with different cancer types, and it is increasingly recognized as a potential therapeutic target in oncology. Here, utilizing core hopping and conformational restriction strategies, we designed and synthesized a series of coumarin analogs that were shown to be potent LSD1 inhibitors in the enzyme assay. Furthermore, several potent compounds were selected to evaluate their antiproliferative activity on A549 cells and MGC-803 cells with high expression of LSD1. Among them, YX10 showed an anticlonogenic effect on A549 cells and MGC-803 cells, with IC50 values of 1.52 ± 0.16 and 0.98 ± 0.18 μM, respectively. Modeling suggested that the inhibitors would bind to the active site of the protein located around the key residues of Asp555 and Lys661. Meanwhile, a preliminary druggability evaluation showed that compound YX10 showed favorable liver microsomal and moderate plasma stability and weak inhibitory activity against cytochrome P450 isoforms at 10 μM. All the results indicated that compound YX10 could represent a promising lead compound for further development.

Abstract P199: Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo

Abstract In an initial safety evaluation in domestic cats suffering from oral squamous cell carcinomas (FOSCC), nine cat subjects were treated IV with 5-6 doses given twice weekly of s50 RNAi feCK2 at either 2 or 20 ug/kg (Cannon et. al., Hu Gene Ther Clin Devel 28(2):80-6, 2017). Treatment was generally well tolerated. In one case, a clear drug-related Grade 3 side effect was rapid tumor death (tumor lysis) resulting in an open wound. This issue has been addressed successfully in separate mice studies with longer, lower-dose SQ regimens. Protein kinase CK2, as a promising target in oncology, plays vital roles in cell growth, proliferation, and suppression of apoptosis (Ahmed et al., Trends Cell Biol 12: 226-30, 2002). Anti-feCK2aa’ is a tumor-targeted 20 nm crystalline capsule bearing single-stranded RNAi oligos against both CK2 catalytic kinase subunits with a ligand-coated shell derived from Tenascin-C (tenfibgen). Tenascin-C is upregulated in multiple solid tumors and is present in tumors throughout their lifecycle. The capsule’s ultrasmall size enables efficient raft-mediated delivery of oligos to the perinuclear space of the target cell, and the ability to reach metastases without reliance on EPR (Unger et. al, Mol Cancer Ther 13:2018-29, 2014). Initial mechanistic work focused on changes in clinically scored IHC of the CK2a subunit in study tumors. In an effort to develop a future working hypothesis, remaining paraffin-embedded tissues were retrospectively examined for both kinase subunits of CK2 as a mixture (CK2aa’), cleaved PARP (CP, apoptosis marker) and Ago2 (critical enzyme required for RNAi drug activity). Previous work in murine models showed variation of Ago2 levels in primary human and xenograft tumors. Further, model xenograft and syngeneic tumors responded with increased Ago2 levels and drug activity upon 10 mg/ml IV priming followed by low-dose SQ treatment (Cancer Res 2016;76(14 Suppl):3746). Using confocal fluorescence microscopy, we observed that decreased post-treatment CK2aa’ and increased CP corresponded positively with clinical tumor responses while non-responders showed an inverse relationship. Examination of arrayed human HNC samples also showed a variation in Ago2 levels but premalignant oral lesions from a human chemoprevention trial did not. These observations suggest a possible future path for feline patient therapy of advanced lesions using Ago2 stratification, potential IV priming and more convenient, longer-term SQ dosing administered by feline caregivers. A path for human therapy may lie through chemoprevention with much to be learned from feline studies. Funding: University of Minnesota Clinical and Translational Sciences Award (1UL1 RR033183-01), NIH SBIR Contract HHSN26120 1300030C, N01-CN-15000, Mod#5, V.A. Merit Review research funds (BX00173 and BX003282) and NCI Award CA150182. Citation Format: Frank G. Ondrey, Gretchen M. Unger, Claire M. Cannon, Janeen H. Trembley, BT Kren, Jaime F. Modiano, Khalil Ahmed. Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P199.

Abstract P238: Derazantinib, an inhibitor of fibroblast growth factor receptors 1-3, synergises with paclitaxel in pre-clinical gastric tumor models

Abstract Background: Derazantinib (DZB) is an oral fibroblast growth factor receptor (FGFR) inhibitor with clinical activity in intrahepatic cholangiocarcinoma. Kinase assays indicate activity against other important targets in oncology, including CSF1R and VEGFR2. We have shown that DZB can inhibit phosphorylation of CSF1R upon ligand stimulation in mouse macrophages ex vivo (GI50=100 nM); suggesting tumor-associated macrophages (TAMs) as an important target for DZB. Paclitaxel also reduces M2-TAM function, suggesting a potential synergy when combined with DZB. DZB monotherapy showed strong efficacy in some gastric (GA) PDX tumor models, so we have investigated the combination of DZB and paclitaxel in several GA-models in vivo. Materials and Methods: Four human tumor GA cell lines were studied in vitro: SNU-16, Fu97, AGS and KATOIII. SNU-16 (FGFR2-fusion) was grown s.c. in Balb/c mice as a xenograft (CDX), and 5 different patient-derived xenografts (PDX) with various FGFR-aberrations (fusion, amplification, over-expression or mutation) were grown in the same host. Mice were treated with different doses of DZB (p.o., qd) alone, and/or with paclitaxel (15 mg/kg, i.v., qw) for 3-4 weeks when tumors were 150 mm3. At the endpoint, tumors were ablated and snap-frozen or paraffin-embedded (FFPE) for western-blot/qPCR or immunohistochemistry (IHC), respectively. Efficacy was summarized as the endpoint dT/C, and the interaction assessed formally as synergy/additivity/antagonism by the Clarke-Combination-Index (CCI). The statistical significance of M2-TAMs was assessed using a one-tailed Fisher contingency test. The tumor models were run by CrownBio Inc, which also provided data on the FGFR-aberrations. Results: In vitro, DZB showed synergy with paclitaxel in SNU-16 and Fu97 models at concentrations known to be achievable in mouse plasma. In vivo, three experiments with the SNU-16 model showed reproducible synergy (mean CCI = -0.64) with the combination causing at least stasis and some complete-regressions. A PD-study after 3-days treatment showed a significant decrease in Ki67 and increase in the M1-TAMs in the combination group. Plasma PK showed no indication of a drug-drug interaction between the two compounds. In the 5 PDX-models, the combination showed synergy in three models and additivity in two. IHC analysis of M2-TAM levels in vehicle-treated mice of all 6 models showed that the two additive models had M2-TAM levels ≤0.8%, while synergy was seen in the 4 models with M2-TAMs of 1.1-8.7%. (p=0.03, using a cut-off of 1%). Conclusions: DZB combined with paclitaxel in vivo showed synergy/additivity in GA-tumor models with FGFR aberrations including FGFR2-fusions/amplifications, and FGFR1-3 over-expression or mutations. Additionally, the number of M2-TAMs may also play a role in sensitivity to the combination, which is consistent with the CSF1R kinase being an important target for DZB. Citation Format: Paul M. McSheehy, Mahmoud El Shemerly, Felix Bachmann, Laurenz Kellenberger, Heidi Lane. Derazantinib, an inhibitor of fibroblast growth factor receptors 1-3, synergises with paclitaxel in pre-clinical gastric tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P238.

Therapeutic Influence on Important Targets Associated with Chronic Inflammation and Oxidative Stress in Cancer Treatment.

Simple SummaryThere is no doubt that the need for new effective methods of cancer treatment remains challenging, as cancer is the second cause of death based on the number of cases in the world. In this review, we investigated the role of one of the leading determinants in the development and progression of various types of cancer—oxidative stress and inflammation, as well as clinical and experimental data from the studies of promising drugs of natural origin, such as flavonoids, that target these stages of oncogenesis. This can all help in the expansion and systematization of the existing knowledge regarding the fight against cancer, the facilitation of the development of effective anti-cancer drugs, and the progression of research in this field, in order to improve the treatment of these disorders.Chronic inflammation and oxidative stress are the interconnected pathological processes, which lead to cancer initiation and progression. The growing level of oxidative and inflammatory damage was shown to increase cancer severity and contribute to tumor spread. The overproduction of reactive oxygen species (ROS), which is associated with the reduced capacity of the endogenous cell defense mechanisms and/or metabolic imbalance, is the main contributor to oxidative stress. An abnormal level of ROS was defined as a predisposing factor for the cell transformation that could trigger pro-oncogenic signaling pathways, induce changes in gene expression, and facilitate accumulation of mutations, DNA damage, and genomic instability. Additionally, the activation of transcription factors caused by a prolonged oxidative stress, including NF-κB, p53, HIF1α, etc., leads to the expression of several genes responsible for inflammation. The resulting hyperactivation of inflammatory mediators, including TNFα, TGF-β, interleukins, and prostaglandins can contribute to the development of neoplasia. Pro-inflammatory cytokines were shown to trigger adaptive reactions and the acquisition of resistance by tumor cells to apoptosis, while promoting proliferation, invasion, and angiogenesis. Moreover, the chronic inflammatory response leads to the excessive production of free radicals, which further aggravate the initiated reactions. This review summarizes the recent data and progress in the discovery of mechanisms that associate oxidative stress and chronic inflammation with cancer onset and metastasis. In addition, the review provides insights for the development of therapeutic approaches and the discovery of natural substances that will be able to simultaneously inhibit several key oncological and inflammation-related targets.

Abstract P250: Evaluation of systemic pan-CLK/DYRK inhibition on organ function and tissue self-renewal

Abstract Proteome diversification through extensive pre-mRNA alternative splicing (AS) is a fundamental aspect of cell fate determination and tissue specialization. The CDC2-like kinases (CLK1-4) and the dual specificity tyrosine phosphorylation regulated kinases (DYRK1-4) have been recognized as participating in signal transduction-dependent alternative splice junction selection through phosphorylation of their substrate proteins- the serine/arginine rich splicing factors (SRSF1-12). The use of CLK/DYRK kinase inhibitors, with various selectivity and potency profiles within the CMGC family, has implicated this regulatory process in both development and disease. Furthermore, an emerging appreciation for the key role of AS in support of tumorigenesis indicates the CLK/DYRK kinases may represent a druggable oncology target class. However, careful evaluation of potential toxicities associated with inhibition of these kinases has not yet been reported. To address this need, the preclinical toxicity profile of SM08502, a potent pan-CLK/DYRK inhibitor was characterized in multiple exploratory and GLP studies performed in rats and cynomolgus monkeys. Daily dosing of SM08502 was generally well-tolerated in both rats and monkeys, with limited target organs of toxicities. Major clinically relevant target organ of toxicities included gastrointestinal (GI), bone marrow, and lymphatic organ toxicities in both rats and monkeys. GI toxicity was characterized as villous blunting, single cell necrosis of the epithelium, hemorrhage, inflammation, and edema. These findings in the GI tract manifested clinically as emesis, inappetence, vomiting, and diarrhea. Microscopic findings suggest a distinct mechanism from cytotoxic agents such as chemotherapy. Morphological evaluation suggests SM08502 primarily affects GI villar epithelial cell dynamics without impairing crypt cell replication or survival. Complete recovery of GI tract findings upon dose holiday was demonstrated in both the rat and the monkey. The SM08503-related hematological findings included decreased in reticulocytes, and leukocytes (primarily monocytes and lymphocytes) in both species. These hematological effects contributed to decreased hematopoietic cellularity (all lineages) in the bone marrow and lymphocyte depletion in lymphatic organs (thymus, spleen, lymph nodes and GALT (gut-associated lymphoid tissue). The nonclinical experience with SM08502 suggests an acceptable safety profile, with adverse events limited to transient reversible GI and hematological findings. Citation Format: Hadi Falahatpisheh, Maryam Dehghani, Jane Morrow, Catherine Tsao, Steven Dorrow, Josh Stewart, Carine Bossard, Brian Eastman, Chi-Ching Mak, Karamjeet Pandher, Michael A. White. Evaluation of systemic pan-CLK/DYRK inhibition on organ function and tissue self-renewal [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P250.

Abstract P131: The role of UDP-6 glucose dehydrogenase (UGDH) in estrogen-mediated phenotypes in both estrogen receptor positive and estrogen receptor negative breast cancer

Abstract A better understanding of pathologic mechanisms underlying breast cancer progression and metastasis is critical to improve current treatment modalities. UDP-6- glucose dehydrogenase (UGDH) is an enzyme that has become a recent oncologic target of interest across various cancer subtypes for its role in increasing the aggressiveness and migratory capacity of tumor cells both in vitro and in vivo. UGDH is ubiquitously expressed and plays a critical role in forming the extracellular matrix (ECM), producing nucleotide sugars, and processing hormones through glucuronidation. For these reasons, investigating the effect of UGDH on breast cancer in the setting of hormonal stimulation is important. We found that knocking down UGDH in estrogen receptor positive (ER+) breast cancer cell lines (MCF7 marco and T47D) and an estrogen receptor negative (ER-) breast cancer cell line (MDA-MB-231) could abrogate estrogen induced migration-specific phenotypes in vitro. Furthermore, we found that knocking down UGDH mitigated estrogen stimulated primary tumor growth in vivo for both MCF7 macro and MDA-MB-231 mammary fat pad tumor models in mice. Thus, we found that UGDH regulates estrogen stimulated migratory phenotypes in both ER+ and ER- breast cancers in vitro and primary tumor growth in vivo. For this reason, UGDH and its associated pathways are promising targets for future drug development and small molecular targets in the treatment of both metastatic ER positive and ER negative breast cancers. Citation Format: Meghan Price, Catherine Lavau, Cesar Baeta, Jovita Byemerwa, Suzanne Wardell, Olivia Brueckner, Debarati Mukherjee, Corinne Haynes, Annee Nguyen, Chingyi Chang, Donald McDonnell, C. Rory Goodwin. The role of UDP-6 glucose dehydrogenase (UGDH) in estrogen-mediated phenotypes in both estrogen receptor positive and estrogen receptor negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P131.

Combinatorial Genetic Uncovers DOCK1/RAC2 As Specific Targets for the Treatment of NPM1;Cohesin Mutated AML

Acute myeloid leukemia (AML) is an aggressive malignancy of the blood and bone marrow resulting from the accumulation of multiple serially acquired mutations. The mutational complexity of the disease makes AML difficult to treat, contributing to a low 5-year survival rate. A better understanding of how different mutations interact with one another to influence disease characteristics is critical and may result in the development of targeted therapies to improve patient outcome. The most common recurrent somatic mutation in AML affects the gene NPM1 and occurs in 25-30% of patients. This mutation results in the aberrant cytoplasmic localization of the protein and is termed NPM1cA (Falini et al. 2005, Cancer Genome Atlas Research Network 2013). NPM1cA is considered to be a driver of AML, however Npm1 cA/+ mice only develop disease after a long latency (median 18 months), suggesting that other cooperating mutations are required for AML development (Vassiliou et al. 2011).Approximately 50% of patients with an NPM1cA mutation also harbor a mutation in one of four members of the cohesin complex (STAG2, SMC3, SMC1A, and RAD21). Mutations in cohesin genes are mutually exclusive and result in haploinsufficiency of the complex. As cohesin is known to regulate gene expression by facilitating promoter-enhancer interactions and three-dimensional genome organization, we wished to determine how cohesin mutation influences AML biology and gene expression in the presence of NPM1cA. We utilized the inducible Npm1 cAflox/+ and Smc3 flox/+ mouse models to examine this genetic interaction. We and others have shown that cohesin mutations result in enhanced hematopoietic stem and progenitor cell (HSPC) self-renewal (Mazumdar et al. 2015, Viny et al. 2015, Mullenders et al. 2015, Galeev et al. 2016, Fisher et al. 2017). Consistent with this, Npm1 cA/+;Smc3 Δ/+ HSPCs show enhanced self-renewal in vitro over HSPCs harboring either single mutation. Despite a shared role in self-renewal, Npm1 cA/+;Smc3 Δ/+mice developed AML with similar latency as Npm1 cA/+ mice. Interestingly, however, Npm1 cA/+;Smc3 Δ/+ HSPCs exhibited dysregulation of a unique set of genes compared to cells from either single mutant, suggesting that the Npm1 cA;cohesin mutational combination uniquely alters the transcriptional environment. Further, Npm1 cA/+;Smc3 Δ/+ leukemias had a completely unique spectrum of acquired mutations compared to Npm1 cA/+ leukemias, suggesting that the addition of Smc3 haploinsufficiency to Npm1 cA/+alters AML evolution and that different driver mutations result in the accumulation of very different somatic mutations.Among the uniquely upregulated genes in Npm1 cA/+;Smc3 Δ/+ HSPCs is Dock1, a guanine nucleotide exchange factor (GEF) for Rac1/2. As high Dock1 expression has been associated with low overall and disease-free survival in multiple cohorts of AML patients (Lee et al. 2017, Zhang et al. 2019), we hypothesized that Dock1 would be a novel target for the treatment of Npm1cA; cohesin mut leukemias. Consistent with this hypothesis, We found that knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis in an Npm1 cA/+;Smc3 Δ/+leukemic line, but not in an Npm1 cAsingle mutant line. Higher Rac activity was also observed in Npm1 cA/+;Smc3 Δ/+ vs. Npm1 cA/+ leukemic lines. We found that DN Rac2 specifically impacted both the growth and apoptosis of an Npm1 cA/+;Smc3 Δ/+line, suggesting that Dock1 functions primarily through Rac2 to regulate survival. Importantly, the Dock1/Rac pathway is specifically targetable in Npm1 cA/+;Smc3 Δ/+ AMLs in vitro and in vivo, as knockdown of Dock1 resulted in prolonged latency in a Npm1 cA/+;Smc3 Δ/+ transplant model and slowed the growth and enhanced apoptosis of an Npm1 cA/+;Smc3 Δ/+, but not an Npm1 cA/+, leukemic line. Further, small molecule inhibitors of Dock and Rac had similar effects. Our results suggest that Dock1/Rac2 represent unique targets for the treatment of patients harboring the NPM1cA;cohesin mutational combination and provide validity to the concept that combinatorial genetics can uncover novel precision oncology targets. DisclosuresVassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy.

Discovery of SY-5609: A Selective, Noncovalent Inhibitor of CDK7.

CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

Oncogenic role of karyopherin α2 (KPNA2) in human tumors: A pan-cancer analysis

BackgroundKPNA2, a nuclear export protein that plays an important role in tumorigenesis, is an emerging hotspot target in oncology. Despite increasing supporting evidence of its importance, no pan-cancer analysis, across multiple databases, is available for in-depth data mining of the gene. MethodsTumor data from both The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were explored to investigate the potential oncogenic roles of KPNA2. Diverse analytical methods were used to gain a full-scale understanding of KPNA2: gene expression, survival situations, genetic mutations, DNA methylation, sites of protein phosphorylation, immunocyte infiltration, and correlative cellular pathways. ResultsKPNA2 is highly expressed in many cancers, and different correlations exist between KPNA2 expression and prognosis of cancer patients. cBioPortal reported that a nonsense mutation of R285* was considered to be the primary tumorigenic genetic alteration to KPNA2 and was found in cases of LUSC, STAD, and CESC. Enhanced phosphorylation of S62 was found in several cancers and the level of infiltration of cancer-associated fibroblasts was found to be linearly correlated with KPNA2 expression levels in ACC, BRCA, MESO, TGCT, THCA, and THYM. Correlations between KPNA2 DNA methylation and the pathogenesis of various tumors in TCGA were further identified. KEGG and GO enrichment analysis identified cell cycle, microtubule binding, and tubulin binding functions for KPNA2. ConclusionThis is the first pan-cancer analysis focusing on KPNA2. It provides a comprehensive understanding about the role of KPNA2 in tumorigenesis and highlights the potential targeted role of KPNA2 for cancer study.

Predictive Ability of Delta Radiomic Texture Features (DRTF) Extracted from Liver Patients Treated With Magnetic Resonance Guided Stereotactic Body Radiotherapy (MRgSBRT)

In the abdomen, real-time MRgSBRT has live motion tracking, high soft tissue contrast, and direct visualization of oncologic targets and organs at risk. Setup images are acquired daily, which generates longitudinal, non-invasive radiographic data points. Our study aimed to identify delta radiomic texture features (DRTF) extracted from daily set up images to predict local progression in liver patients treated with MRgSBRT.This work included a prospective analysis of an IRB-approved database of 20 patients with liver lesions treated by MRgSBRT. The gross tumor volume was identified on daily low field strength (0.35T) setup images. MR images were pre-processed using histogram equalization to re-bin the gray level intensities to 64 levels and 39 second-order DRTFs extracted. DRTFs were calculated as the difference between values on the initial set up scan and after intervals of BED 20 Gy (α/β = 10) were delivered. The Gini Index was used to rank features in order of predictive importance during training of a random forest model while using the delta radiomics texture features to predict local control (LC) at 1 year. Leave-one-out (LOO) analysis was performed using the top two features.Lesions were treated to a median dose of 50 Gy in 5 fractions, or BED 20 Gy per fraction. The top two features identified after BED 20 Gy (1 fraction) was delivered were gray level co-occurrence matrix features (GLCM) and gray level size zone matrix (GLSZM) based Large Zone Emphasis. The model generated an ROC curve with AUC = 0.9011 [0.752 - 1] using LOO analysis. The same two features were selected as predictive after delivery of BED 40 Gy and LOO analysis resulted in a ROC curve with AUC = 0.6868 [0.3957 - 0.9779].DRTFs extracted from daily setup MR images after a single fraction of MRgSBRT were predictive of LC in this cohort. Additional studies are needed to evaluate these features as they may identify patients with radioresistant disease and provide an opportunity for physicians to dose escalate.

Breast Cancer Stage Is Associated With Exceeding Target Price in the Oncology Care Model.

To explore mean difference between Oncology Care Model (OCM) total costs and target price among breast cancer episodes by stage under the Centers for Medicare and Medicaid Services OCM payment methodology. Breast cancer episodes from OCM performance period 1-4 reconciliation reports (July 1, 2016-July 1, 2018) were linked with health record data from a large, academic medical center. Demographics, total cost of care (TCOC), and target price were measured by stage. Adjusted differences between TCOC and target price were compared across cancer stage using multivariable linear regression. A total of 539 episodes were evaluated from 252 unique patients with breast cancer, of which 235 (44%) were stage I, 124 (23%) stage II, 33 (6%) stage III, and 147 (27%) stage IV. About 37% of episodes exceeded target price. Mean differences from target price were -$1,782, $2,246, -$6,032, and $11,379 all in US dollars (USD) for stages I through IV, respectively. Stage IV episodes had highest mean TCOC ($44,210 USD) and mean target price ($32,831 USD) but also had higher rates of chemotherapy, inpatient admission, and novel therapy use. After adjusting for covariates, stage IV and ≥ 65-year-old patients had the highest mean difference from target price ($17,175 USD; 95% CI, $12,452 to $21,898 USD). Breast cancer episodes in older women with distant metastases most frequently exceeded target price, suggesting that target price did not adequately account for complexity of metastatic cancers. A metastatic adjustment introduced in PP7 represents a promising advancement in the target price methodology and an impact evaluation will be needed.