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Cushing's disease (CD) is a serious endocrine disorder caused by dysregulated adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery, which is followed by disease remission. Current surgical remission rates reported range from 47–85% depending on several remission criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies makes CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of human relevant model systems that recapitulate the cellular composition of pituitary adenomas. Human pituitary adenoma tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). hPITOs generated from corticotroph, lactotroph, gonadotroph and somatotroph adenomas exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of pituitary adenoma specific markers using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each adenoma subtype. Generation of induced pluripotent stem cells (iPSCs) from patients carrying germline mutations relevant to CD exhibited dysregulated cell lineage commitment. The human pituitary adenoma organoids represent a novel approach in how we model complex pathologies in CD patients that may enable more effective personalized medicine for these patients.

Glioblastoma multiforme (GBM) is an aggressive stage IV brain cancer that is difficult to detect, typically inoperable, and associated with a poor prognosis. Challenges in targeting GBM arise from the involvement of various pro-survival pathways, being comprised of four different cellular states, and displaying heterogeneity between each tumor. Marketed FDA-approved drugs for GBM have demonstrated limited efficacy, toxicity issues, resistance, and poor BBB penetrance. Further probing into pathway breakdowns led to the discovery of the DYRKs (dual-specificity tyrosine-regulated kinase), CLKs (CDC-like kinases), PDGFRA/B (platelet-derived growth factor receptor), and PI3Kα (phosphoinositide 3-kinase) involvement and overexpression. The upregulation of these kinases are found to be prominently involved in GBM cell proliferation, survival, replication, aggression, metastasis, and resistance. Herein, we believe that the development of pleiotropic competitive inhibitors for the treatment of GBM could be a promising therapeutic option based on literature precedence and current data from research. DYR726, a water-soluble, pleiotropic inhibitor [IC50 PDGFRA/B/PI3Kα/CLk2/3DYRK1a/2/3 ( 53.0/48.0/6.2/15.13/137/4/38.6/11.2 nM)] has demonstrated in vitro cellular efficacy across various GBM cell lines. When treated in non-cancerous cell lines, DYR726 displayed an optimal therapeutic window, which is not observed in any glioma drugs currently in clinical trials. At 1µM concentrations, complete dissociation of the formation of neural stem cells and neurosphere growth in glioma cells are observed. Reduction in metastasis and proliferation are found when treated with DYR726 over three weeks. In vitro efficacy against current approved FDA treatment options and clinical candidates has been extensively tested across numerous GBM cell lines. When benched against one of the best PI3K kinase inhibitors in the clinic, Buparlisib, DYR726 was comparable, indicating a disruption in the RTK signaling survival pathway that is commonly associated with GBM. DYR726 performed favorably against Avapritinib (clinical trials for gliomas are beginning at Stanford) and when treated against FDA-approved combination treatment Dabrafenib+Trametinib (for 2% of glioma patients), it performed identically, suggesting that DYR726 can inhibit the cells at the same rate as the new FDA drug. Within patient-derived cell lines, it behaves identically to Buparlisib, and DYR726 has been shown to outperform AstraZenecas’ Osimertinib (which was significantly less potent) and Tafinlars’ Dabrafenib (displayed no effect). Based on the significance of efficacy, this series is on the CRUK Glasgow Cancer Center grant renewal as a potential Phase 0 within the next five years. Citation Format: Alessandra Fistrovich, Vasudah Tandon, Carly Cabel, Laura Basantes, Curtis Thorne, William Montfort, Sourav Banerjee, Christopher Hulme. The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3100.

The purpose of this study is to determine the function of CLK3 in the colonic epithelium and colon cancer. The intestinal epithelium undergoes constant renewal and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways including cellular proliferation. Our lab previously performed an siRNA screen to investigate hundreds of kinases to identify candidates which could interact with GSK3. Among the over 500 kinases that were tested, a protein kinase called CDC-like kinase 3 (CLK3) was identified as the strongest interaction with GSK3. We found that inhibition of GSK3 and CLK3 synergized to kill cancer cells. This led us to focus on CLK3 as a potential therapeutic target kinase in colon cancer. CLK3 is part of a four-member family of CDC-like kinases involved in pre-mRNA splicing. They are dual-specificity kinases, meaning they can target both tyrosine and serine/threonine substrates. CLK3 is also a poor prognostic marker in colon cancer, with high expression of CLK3 leading to poor outcome of disease. In human colonic epithelial cells, we show that loss of CLK3 dampens Wnt response. This data, along with the strong genetic interaction with GSK3, suggests a role of CLK3 in the Wnt pathway. Because Wnt signaling is higher in the stem cell region of the crypt, and CLK3 shows a role in Wnt activity in human colonic epithelial cells, we hypothesize that CLK3 regulates mRNA splicing to promote Wnt signaling and stemness in colonic progenitor cells and carcinogenesis. We are currently performing transcriptomics and phosphoproteomics in CLK3 CRISPR knockout cells to identify an mRNA-splicing and phosphorylation signature. We have found that the induction of Wnt-related pathway genes are lost in CLK3 knockout cells. Additionally, we are categorizing CLK3-dependent pre-mRNAs splicing events into functional groups controlling cell fate and validating in patient-derived normal and tumor organoid models. This work highlights CLK3 as a critical regulator of gut stem cells and potentially colorectal cancer stem cells. Citation Format: Carly Cabel, Curtis Thorne. Characterizing CLK3 in the colonic epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 165.

Background: The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC). Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify regulatory mechanisms underlying APC mutation-negative (APCmut-) CRCs. Methods: We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs. We also constructed and compared regulatory networks between APCmut- and APCmut+ CRCs by integrating transcription factor (TF) binding motifs, protein-protein interactions, and gene co-expression using the algorithms PANDA and LIONESS. Single sample networks were used to identify each tumor’s contribution to the differences in transcriptional regulation observed between the APCmut- and APCmut+ groups. Results: APCmut- CRC expression profiles clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. Consistent with this, APCmut-CRC regulatory networks showed strong activation of immune pathways relative to APCmut+ CRCs. The second most prominently targeted pathway was RAS signaling, with the TFs TCF15 and RUNX2 regulating key pathway members including RAF1, GEFs, and GAPs. Single sample networks revealed that tumors with the strongest transcriptional regulation of RAS genes tend to lack a KRAS mutation and exhibit a slight skew towards earlier onset. Conclusions: APCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways. APCmut- tumors exhibit increased regulation of RAS pathway genes compared to APCmut+ tumors. The samples contributing most to this regulatory signature tend to lack a KRAS mutation, suggesting that APCmut- tumors have developed ways to modulate RAS pathway activity through non-mutational mechanisms. Focal emphasis on the RAF1 gene suggests these tumors may be more vulnerable to inhibition of the downstream MAPK pathway. Network analysis implicates TCF15 and RUNX2 as regulators of RAS pathway genes, suggesting a possible WNT-RAS crosstalk occurring in APCmut- CRC that warrants further investigation. Citation Format: Megha Padi, Dante Bellomo, Adam Grant, Rosa M. Xicola, Curtis Thorne, Bodour Salhia, Xavier Llor, Nathan Ellis. Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 484.