Multiple Kinase Inhibitor Research Articles

Overall survival of patients with KIT-mutant metastatic GIST in the era of multiple kinase inhibitor availability

PurposeThe prognosis of patients with metastatic GIST and imatinib-sensitive primary mutations has significantly improved. However, limited data are available to inform patients about outcomes across different lines of treatment. This retrospective analysis aims to evaluate patient outcomes at a large German GIST referral center over the past 15 years.Patients and methodsOverall survival (OS) and progression-free survival (PFS) were analyzed in patients with metastatic GIST, with diagnosis of metastases between 2008 and 2021, when at least three lines of treatment were available in Germany (n = 174).ResultsThe median overall survival far exceeds historical data for patients with primary exon 11 and exon 9 mutations (median OS in palliative treatment with imatinib: 7.1 years; median OS in second-line palliative treatment with sunitinib: 2.9 years; median OS in third-line palliative treatment with regorafenib: 1.9 years). Among those patients who received palliative imatinib treatment, no significant difference in median OS survival was observed between those who had received perioperative imatinib for localized disease and those who did not. Furthermore, the location of metastases significantly impacted survival, whereas the time between the initial diagnosis and the diagnosis of metastases had no significant effect on survival.ConclusionIn conclusion, this study provides a novel, real-world reference for survival outcomes in patients with metastatic GIST.

Identification of BAY61-3606 Derivatives With Improved Activity in Splicing Modulation That Induces Inclusion of Cassette Exons Similar to the Splicing Factor 3B Subunit 1 Mutation.

Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities.

Amcasertib Increases Apoptosis While Decreasing Invasive and Migrating Abilities in Breast Cancer Stem Cells

Objective: A relationship exists between breast cancer stem cells (BCSCs) and the chemo-resistance and recurrence of aggressive breast tumors. Amcasertib is a small chemical compound and multiple kinase inhibitor that inhibits downstream Nanog and other cancer stem signaling pathways in cancer stem cells by targeting several serine-threonine kinases. In this study, we aimed to investigate the cytotoxicity and anticancer effects of Amcasertib on BCSCs, gaining insight into the targetability of BCSCs. Method: We used the combined xCELLigence-Real-Time Cell Analyzer (RTCA) equipment to analyze cytotoxicity and cell proliferation. We detected the IC50 dosages of Amcasertib at 24, 48, and 72 hours and examined its effects on apoptosis, cell cycle, invasion, and migration over 48 hours. We used flow cytometry for assays of apoptosis and cell cycle, and the CytoSelect 96-well Cell Migration and Invasion Assay Kit for evaluating invasion and migration. Results: Our results showed that Amcasertib has cytotoxic properties, with an IC50 dosage of 1.9 μM at the 48th hour. In addition, Amcasertib significantly induced apoptosis in BCSCs, despite not affecting the cell cycle. Moreover, Amcasertib decreased BCSCs’ invasion and migratory properties, part of epithelial-mesenchymal transition (EMT). Conclusion: In conclusion, our findings provide crucial information for understanding the potential of Amcasertib in targeting BCSCs. In addition, we suggest that Amcasertib could be a beneficial drug for breast cancer treatment by targeting BCSCs.

Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer

BackgroundTreatment with regorafenib, a multiple-kinase inhibitor, to manage metastatic colorectal cancers (mCRCs) shows a modest improvement in overall survival but is associated with severe toxicities. Thus, to reduce regorafenib-induced toxicity, we used regorafenib at low concentration along with a dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition to enhance antitumor activity. The therapeutic efficacy and safety of the combination treatment was evaluated with CRC models.MethodsThe cytotoxicity of JAK/HDACi, regorafenib, and their combination were tested with normal colonic and CRC cells exhibiting various genetic backgrounds. Kinomic, ATAC-seq, RNA-seq, cell cycle, and apoptosis analyses were performed to evaluate the cellular functions/molecular alterations affected by the combination. Efficacy of the combination was assessed using patient-derived xenograft (PDX) and experimental metastasis models of CRC. To evaluate the interplay between tumor, its microenvironment, and modulation of immune response, MC38 syngeneic mice were utilized.ResultsThe combination therapy decreased cell viability; phosphorylation of JAKs, STAT3, EGFR, and other key kinases; and inhibited deacetylation of histone H3K9, H4K8, and alpha tubulin proteins. It induced cell cycle arrest at G0-G1 phase and apoptosis of CRC cells. Whole transcriptomic analysis showed that combination treatment modulated molecules involved in apoptosis, extracellular matrix-receptor interaction, and focal adhesion pathways. It synergistically reduces PDX tumor growth and experimental metastasis, and, in a syngeneic mouse model, the treatment enhances the antitumor immune response as evidenced by higher infiltration of CD45 and cytotoxic cells. Pharmacokinetic studies showed that combination increased the bioavailability of regorafenib.ConclusionsThe combination treatment was more effective than with regorafenib or JAK/HDACi alone, and had minimal toxicity. A clinical trial to evaluate this combination for treatment of mCRCs is warranted.

Pembrolizumab-induced resolving papulosquamous eruption: An enigmatic presentation in a patient with renal cell carcinoma

Lenvatinib, a multiple tyrosine receptor kinase inhibitor, and pembrolizumab, an anti-programmed cell death protein 1 humanized antibody, both are indicated in the treatment of renal cell carcinoma (RCC). Both these agents rarely produce cutaneous adverse events. In this instance, a RCC patient who received both medications and experienced a papulosquamous eruption as an uncommon cutaneous side effect of both medications is being reported. The potential drug triggers preceded the symptom onset and the observed symptom resolved on discontinuation of pembrolizumab.

A phase I/II study of C019199 in combination with sintilimab in advanced malignancy.

e15110 Background: C019199 is an oral CSF-1R/DDRs/VEGFR2 multiple kinase inhibitor and modulates the immune micro-environment of tumor cells. Previous phase I study of C019199 as a mono-therapy for advanced solid tumors has shown promising safety and anti-tumor activity. This phase I/II study is to evaluate the safety and efficacy of the combination of C019199 and Sintilimab in solid tumors. Methods: An open label, multicenter phase I/II study of C019199 combined with Sintilimab is currently enrolling (NCT06220318). Eligible subjects (age of ≥18 years and <76 years) with histologically or cytologically confirmed relapsed, refractory, or progressive metastatic solid tumors will be enrolled in. This trial consists of 2 parts: a phase I dose escalation study using a standard 3+3 design with 3 doses of C019199 (100 mg/qd, 200 mg/qd, 300 mg/qd) combined with a fixed dose of Sintilimab (200 mg, iv, q3w) to determine the maximum tolerated dose (MTD) and recommended phase Ⅱ dose (PR2D); A phase II study is planned to enroll about 20 patients in each tumor type, including but not limited to the following: colorectal cancer, gastric cancer, esophageal squamous cell carcinoma, pancreatic cancer, triple negative breast cancer, head and neck squamous cell carcinoma. Results: A total of 10 patients with advanced solid tumors were enrolled in the phase I study from Jul 20, 2023 to Nov 14, 2023. Median age was 54 (range, 37-71), with 6 males and 4 females. Tumor types were colorectal cancer (7 pts), non-small cell lung cancer (2 pts) and cervical cancer (1 pts), with a median 3 prior systemic therapies (range, 1-7). As of Jan 15, 2024, we have assessed safety in all pts and efficacy in pts with at least one tumor assessment result. No dose-limiting toxicities (DLT) observed in the dose range of 100 mg/qd~300 mg/qd and the MTD has not been reached. The most frequently reported TRAEs were aspartate aminotransferase increased (80%), γ-glutamyltransferase increased (70%), proteinuria (70%), creatine kinase-MB increased (70%), alkaline phosphatase increased (60%). Grade ≥3 TRAEs were hypertension (30%), proteinuria (10%), immune hepatitis (10%), diarrhea (10%). Among 7 colorectal cancer patients, 1 subject had partial responds (PR) and 6 had stable disease (SD) as the best responds with ORR of 14.3%, DCR of 100%. Four subjects were still on treatment. Median PFS was not reached (>3month). Two patients with non-small cell lung cancer and 1 patient with cervical cancer achieved the best response of SD, with a DCR of 100%. Phase II study is ongoing. Recommended dose is 200 mg/qd as the RP2D in selected solid tumors. Conclusions: The combination of C019199 and Sintilimab has shown good overall safety and tolerability. No DLT was observed in the dose range of 100 mg/qd ~ 300 mg/qd. Preliminary anti-tumor activity was observed in patients with advanced solid tumors. Phase II study is ongoing to accumulate more clinical data. Clinical trial information: NCT06220318 .

Beyond Chemoimmunotherapy in Advanced Non-Small Cell Lung Cancer: New Frontiers, New Challenges.

Chemoimmunotherapy is currently the preferred first-line treatment option for the majority of patients with advanced non-small cell lung cancer without driver genetic alterations. Most of these patients, however, will experience disease progression within the first year after treatment initiation and both patients and their physicians will be confronted with the dilemma of the optimal second-line treatment. Identification of molecular targets, such as KRASG12C, BRAFV600X, METexon14, and human epidermal growth factor receptor 2 mutations, and RET rearrangements offer therapeutic opportunities in pretreated patients with corresponding alterations. For those tumors that do not harbor oncogenic drivers, second-line treatment with docetaxel remains the current standard of care despite modest efficacy. Strategies to challenge docetaxel include the combination of immune checkpoint inhibitors (ICIs) with tyrosine inhibitors of multiple kinases or with DNA damage response inhibitors, antibody-drug conjugates, and locoregional treatments for oligoprogressive disease. Next-generation immunotherapy strategies, such as T-cell engagers, immune-mobilizing monoclonal T-cell receptors, chimeric antigen receptor cell therapy, tumor infiltrating lymphocytes, and T-cell receptor cell therapy are being currently investigated in the quest to reverse resistance to ICIs. Importantly, the advent of these new agents heralds a novel spectrum of toxicities that require both the physician's and the patient's education. Herein, we review current and future strategies aiming to outperform docetaxel after chemoimmunotherapy failure, and we provide practical information on how to best communicate to our patients the unique toxicity aspects associated with immunotherapy.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

Deep PIM kinase substrate profiling reveals new rational cotherapeutic strategies for acute myeloid leukemia

AbstractProvirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor–responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor–resistant disease that limits durable responses in malignant disease.

Abstract RF01-07: The efficacy and safety of tinengotinib in patients with advanced or metastatic HR+/HER2- breast cancer or TNBC

Abstract Background: Tinengotinib is a novel multiple kinase inhibitor that strongly inhibits Aurora A/B, FGFR1/2/3, VEGFRs, JAK1/2, and CSF1R. Here we present the preliminary safety, pharmacokinetic and efficacy data of tinengotinib in patients (pts) with hormone receptor positive (HR+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer (BC) and triple-negative BC (TNBC) from Phase I TT420X2101 (NCT03654547) and Phase Ib/II TT420X1103 (NCT04742959) trials. Methods: Eligible pts with HR+/HER2- BC or TNBC who have no available standard therapeutic treatment options were enrolled to the TT420X2101 and TT420X1103 trials. Tinengotinib was given as monotherapy at different dose levels (5 mg QD, 10 mg QD and 12 mg QD); different dosing schedule (6 mg BID); and as combination (tinengotinib 8 mg QD and Abraxane 100 mg/m2). Recruitment spanned from January 2019 to March 2023. Results: As of June 2023, 36 pts with metastatic BC were treated: 30 with tinengotinib monotherapy at dose levels of 5 mg QD (n=1), 8 mg QD (n=4), 10 mg QD (n=5), 12 mg QD (n=18), 6 mg BID (n=2) and 6 with tinengotinib 8 mg QD in combination with nab-paclitaxel 100 mg/m2. Median age 51 years (range 24-75), ECOG PS 1 in 83.3% of pts, median lines of prior therapy were 5, and 77.8% of pts had prior taxanes. Of 30 pts receiving tinengotinib monotherapy, treatment related adverse events (TRAEs) were reported in 26 (86.7%) pts. 43.4% were Grade (G) 1-2, 43.3% were G3. No G4 or G5 TRAEs were reported. Common TRAEs (≥20%) of tinengotinib monotherapy were hypertension (60.0%), stomatitis (50.0%), palmar-plantar erythrodysesthesia syndrome (46.7%) and diarrhea (20.0%). All six (100%) pts receiving tinengotinib in combination with nab-paclitaxel experienced TRAEs, 16.7% were G1, 66.7% were G3, no G4 TRAEs, and one pt had G5 (pulmonary hemorrhage). Common TRAEs (≥20%) of tinengotinib in combination with nab-paclitaxel were neutrophil count decreased/neutropenia (50.0%), stomatitis (50.0%), hypertension (33.3%), hyponatremia (33.3%), hypokalemia (33.3%), and nausea (33.3%). Twenty-eight pts receiving tinengotinib monotherapy were efficacy evaluable. 11 pts with HR+/HER2- BC achieved objective response rate (ORR) of 45.5%, clinical benefit rate (CBR, CR+PR+SD ≥ 24 weeks) of 54.5% and median progression-free survival (mPFS) of 5.55 (95% CI 1.97-6.18) months. Partial responses were seen in 3 pts with HER2-zero (n=5) and 2 pts with HER2 low (1+/2+) pts (n=6), respectively. 17 pts with TNBC had ORR of 23.5%, CBR of 29.4% and mPFS of 2.73 (95% CI 1.68-6.41) months. In 6 pts treated with tinengotinib in combination with nab-paclitaxel, one out of 2 pts with HR+/HER2- BC achieved PR for 13 weeks as of data cutoff; two out of 4 pts with TNBC had SD. No significant difference in exposure was observed between tinengotinib monotherapy and tinengotinib in combination with nab-paclitaxel. Conclusions: Tinengotinib for the treatment of HR+HER2- BC or TNBC, whether as monotherapy or in combination with nab-paclitaxel, had manageable side effects. Tinengotinib has shown promising clinical benefit in heavily pre-treated pts with refractory HR+HER2- BC or TNBC. Clinical benefit was similar across the subgroups of pts with HR+ HER2-zero and HR+ HER2 low disease. Citation Format: Piha-Paul Sarina, Binghe Xu, Ying Fan, Yuan Yuan, Sayeh Lavasani, Joanne Mortimer, Sanjay Goel, Apostolia Tsimberidou, Nuhad Ibrahim, Sausan Abouharb, Carlos Barcenas, Adaeze lheme, Daniel Karp, Jordi Rodon Ahnert, Ecaterina Dumbrava, Jean Fan, Peng Peng, Caixia Sun, Hui Wang, Katie Hennessy, Ximei Fu, Ruolan Xu, Shumao Ni, Frank Wu, Funda Meric-Bernstam. The efficacy and safety of tinengotinib in patients with advanced or metastatic HR+/HER2- breast cancer or TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-07.

Abstract 603: BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models

Abstract Introduction: Immuno-suppressive micro-environment and abnormal signaling pathways are crucial for the proliferation of tumor cells. Hematopoietic progenitor kinase 1 (HPK1) inhibitor plays important roles in activation of exhausted T cells and enhancement of anti-tumor immune responses. VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family, and is reported to be essential for Wnt signaling and colorectal cancer (CRC) proliferation and progression. Blockade of critical kinases in multiple signaling pathways inhibits the proliferation, invasion and metastasis of cancer cells. Therefore, intergradation of these functions in a small molecule serves as the next generation of immune-oncology small molecules for cancer therapy. Methods: BB5523 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. CDX and syngeneic tumor models in mice were conducted to evaluate the in vivo efficacy of BB5523 as a single agent. Results: BB5523 shows high inhibition potency on HPK1 with good selectivity against other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 kinase activity (IC50 = 3.3 nM) by BB5523 strongly suppresses the phosphorylation of the downstream biomarker protein SLP-76 (IC50 = 30 nM); and boosts the IL-2 secretion in purified human T cells and human peripheral blood mononuclear cells. BB5523 also shows good activities on VEGFR2 (IC50 = 1.6 nM) and TNIK (IC50 = 0.7 nM) which are crucial for the proliferation of cancer cells. In in vitro experiments, BB5523 significantly inhibits the proliferation of various tumor cell lines at sub-micromolar concentrations. PK profile of BB5523 shows good oral bioavailability (F > 80%). In preliminary safety evaluation, no abnormal symptoms were observed in mice dosed up to 100 mpk for 14 days. BB5523 showed significant tumor-growth inhibition efficacy in TE-8, JIMT-1, B16-F10 and MC38 tumor models. Conclusion: As a potent and oral available small molecule kinase inhibitor, BB5523 boosts anti-tumor immunity through HPK1 inhibition, while also blocking crucial signaling pathways in tumor micro-environment. BB5523 shows promising in vitro and in vivo efficacy with a well-tolerated safety profile. The preclinical study of BB5523 is in progress. Citation Format: Gongping Duan, Min Li, Puyong Zhang, Wei Hua, Enlun Hao, Shipeng Wang, Xiaoyi Ma, Junheng Wang, Lijie Wei, Xingmin Zhang. BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 603.

Abstract 585: Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model

Abstract Purpose: In the United States, colorectal cancer (CRC) is the second leading cause of cancer mortality. Metastatic CRC (mCRC) refractory to traditional chemotherapy is managed with regorafenib, a multiple-kinase inhibitor. However, patients show only a modest improvement in overall survival but experience high drug toxicity, adverse side effects, and poor tolerability. Thus, to reduce regorafenib-induced toxicity and to enhance antitumor activity, we combined it with a hybrid/dual JAK/HDAC small-molecule inhibitor (JAK/HDACi) to leverage the advantages of both JAK and HDAC inhibition in a single agent. With syngeneic mice, the efficacy and impact on immunomodulation of this drug combination was assessed. Methods: To assess effects of the agents, C57BL/6 immunocompetent mice were injected with MC38 murine CRC cells and treated with regorafenib (6mg/kg body wt) or JAK/HDACi (30mg/kg body wt), and their combination every third day for 21 days. Upon completion of the experiment, the tumors were harvested and processed for RNA and protein expression profiles. The effect of drug treatment on immune response was analyzed by nCounter Gene Expression assays (NanoString Technologies). Cytokines were measured in serum using the MesoScale Discovery mouse V-Plex Proinflammatory Panel kit. For pharmacokinetic studies, plasma samples were assayed from C57BL/6 treated with drugs after 1, 7, 21, and 48 hrs of treatment. Results: The combination treatment significantly reduced tumor growth (volume and weight),relative to the vehicle control or single treatments. Gene expression showed higher CD45 abundance score in the combination treatment. This is an exciting observation, as CD45 has phosphatase activity and dephosphorylates key targets of the drugs used in the study. Additionally, after treatment with the combination, there was higher expression of Gzmb and Gzme and lower expression of Cx3cr1, indicating enhanced immuno modulation relative to regorafenib alone. We confirmed these findings by immunoprofiling of tumors. Higher CD45 staining, noted in tumors of mice treated with the combination, corroborated the gene expression results. Pronounced CD8 T lymphocytes infiltration was observed in combination, compared to regorafenib alone. Reduction of proinflammatory cytokine, TNF-α was noted in the combination group. This is an interesting observation as CD45 negatively regulates TNF, and combination treated mice had highest number of CD45 positive cells. Pharmacokinetic studies showed that the bioavailability of regorafenib was elevated after combination treatment relative to single-agent treatment. Conclusions: Relative to the single agents, the combination of regorafenib with JAK/HDACi was more effective, with an elevated antitumor immune response and with sustained inhibition of tumor growth. A clinical trial to evaluate this combination for the treatment of mCRCs is warranted. Citation Format: Prachi Bajpai, Farrukh Afaq, Sameer Al Diffalha, Sumit Agarwal, Hyung Gyoon Kim, Dennis Otali, Sooryanarayana Varambally, Ashish Manne, Ravi Paluri, Moh’d Khushman, Upender Manne. Regorafenib antitumor immune response is enhanced by a novel drug combination in CRC syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 585.

Abstract 529: Investigating the synergistic effect of milciclib and PD0325901 in colorectal cancer therapy

Abstract Milciclib, characterized as a potent nanomolar inhibitor of multiple cyclin-dependent kinases, has recently emerged as a promising therapeutic agent in cancer treatment, particularly with its additional role as a spliceosome modulator. Our recent studies employing RNA sequencing technology revealed a comprehensive profile of Milciclib-induced transcriptional changes in colorectal cancer cells, identifying several key regulated pathways. In light of these findings, we explored a novel therapeutic strategy: combining Milciclib with specific inhibitors targeting pathways upregulated by Milciclib treatment. This hypothesis is based on the rationale that such a combination could exert a synergistic effect. We have assessed the efficacy of this combinational therapy and found that the combination of Milciclib and PD0325901 greatly potentiates the killing of colorectal cancer cells versus either treatment alone. PD0325901 has been extensively studied for its efficacy in targeting the MEK/ERK signaling pathway, a critical pathway in many cancers, including colorectal cancer. To further characterize the underlying molecular mechanisms contributing to the observed synergistic effect, we applied quantitative proteomic analysis of 8107 proteins in human colorectal cancer cells with the treatment of different doses of Milciclib and PD0325901 administered alone, or in combination. Our results demonstrate 49 proteins were significantly decreased in a dose-dependent manner, and that many of these proteins are key players regulating pathways associated with the cell cycle, apoptosis, gene expression, metabolism, WNT signaling, and the MEK/ERK signaling pathway. The discovery of the down-regulation of these proteins and pathways after treatment provides a molecular basis to elucidate the synergistic mechanism underlying Milciclib and PD0325901 combination therapy. In conclusion, our study establishes the combinatorial use of Milciclib and PD0325901 as a potential breakthrough in the treatment of colorectal cancer, offering a promising prospect for enhancing future patient outcomes. Citation Format: Xinyu Pei, Anh Nguyen, Hannah Neiger, Wenjia Wang, William Bautista, Lily Wang, Alvan Cai, Valerie A. Gerriets, John K. Cusick, Yihui Shi. Investigating the synergistic effect of milciclib and PD0325901 in colorectal cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 529.

Comparative analysis of basal and etoposide-induced alterations in gene expression by DNA-PKcs kinase activity.

Background: Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it also has roles in modulating gene expression and thus, the overall cellular response to DNA damage. Methods: Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Results: Downregulated genes were mostly involved in protein, sugar, and nucleic acid biosynthesis pathways in both genotypes, but enriched biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Finally, we have used qPCR to validate selected genes among the differentially regulated pathways to validate RNA sequence data. Conclusion: Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trials as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

Synthesis of Sorafenib-Ruthenium Complexes, Investigation of Biological Activities and Applications in Drug Delivery Systems as an Anticancer Agent.

Sorafenib, a multiple kinase inhibitor, is widely used as a first-line treatment for hepatocellular carcinoma. However, there is a need for more effective alternatives when sorafenib proves insufficient. In this study, we aimed to design a structure that surpasses sorafenib's efficacy, leading us to synthesize sorafenib-ruthenium complexes for the first time and investigate their properties. Our results indicate that the sorafenib-ruthenium complexes exhibit superior epidermal growth factor receptor (EGFR) inhibition compared to sorafenib alone. Interestingly, among these complexes, Ru3S demonstrated high activity against various cancer cell lines including sorafenib-resistant HepG2 cells while exhibiting significantly lower cytotoxicity than sorafenib in healthy cell lines. Further evaluation of cell cycle, cell apoptosis, and antiangiogenic effects, molecular docking, and molecular dynamics studies revealed that Ru3S holds great potential as a drug candidate. Additionally, when free Ru3S was encapsulated into polymeric micelles M1, enhanced cytotoxicity on HepG2 cells was observed. Collectively, these findings position Ru3S as a promising candidate for EGFR inhibition and warrant further exploration for drug development purposes.

Electrochemical Determination of Anti-Cancer Drug Pazopanib with High Selectivity and Sensitivity Using Molecularly Imprinted Polymer-Modified Glassy Carbon Electrode

Pazopanib (PZB) is a multiple kinase inhibitor used for the treatment of advanced renal cell carcinoma and soft tissue sarcoma. This work focuses on achieving high selectivity and sensitivity for the determination of PZB using a molecularly imprinted polymer (MIP)-based electrochemical sensor. The MIP-based sensor was fabricated by thermal polymerization (TP) directly on a glassy carbon electrode (GCE). The electrochemical response of the 4-ABA/PZB@MIP/GCE sensor was investigated using differential pulse voltammetry (DPV). The characterization of the sensor in terms of morphology and electrochemistry was performed using scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The 4-ABA/PZB@MIP/GCE sensor exhibited a linear response ranging from 1.0 × 10–13 M to 1.0 × 10–12 M with a limit of detection (LOD) and limit of quantification (LOQ) of 1.04 × 10–14 M and 3.48 × 10–14 M, respectively. The applicability of the sensor was evaluated by determining commercial samples of human serum and tablets, and good recoveries were obtained. The results showed that the sensor could identify PZB, compared to structurally analogous drugs such as axitinib, nilotinib, and erlotinib. The interfering substances commonly found in biological fluids were investigated. Finally, the sensor design was validated using a non-imprinted polymer-based GCE.

New 2-oxoindole derivatives as multiple PDGFRα/ß and VEGFR-2 tyrosine kinase inhibitors

Two new series of N-aryl acetamides 6a-o and benzyloxy benzylidenes 9a-p based 2-oxoindole derivatives were designed as potent antiproliferative multiple kinase inhibitors. The results of one-dose NCI antiproliferative screening for compounds 6a-o and 9a-p elucidated that the most promising antiproliferative scaffolds were 6f and 9f, which underwent five-dose testing. Notably, the amido congener 6f was the most potent derivative towards pancreatic ductal adenocarcinoma MDA-PATC53 and PL45 cell lines (IC50 = 1.73 µM and 2.40 µM, respectively), and the benzyloxy derivative 9f was the next potent one with IC50 values of 2.85 µM and 2.96 µM, respectively. Both compounds 6f and 9f demonstrated a favorable safety profile when tested against normal prostate epithelial cells (RWPE-1). Additionally, compound 6f displayed exceptional selectivity as a multiple kinase inhibitor, particularly targeting PDGFRα, PDGFRβ, and VEGFR-2 kinases, with IC50 values of 7.41 nM, 6.18 nM, and 7.49 nM, respectively. In contrast, the reference compound Sunitinib exhibited IC50 values of 43.88 nM, 2.13 nM, and 78.46 nM against the same kinases. The derivative 9f followed closely, with IC50 values of 9.9 nM, 6.62 nM, and 22.21 nM for the respective kinases. Both 6f and 9f disrupt the G2/M cell cycle transition by upregulating p21 and reducing CDK1 and cyclin B1 mRNA levels. The interplay between targeted kinases and these cell cycle regulators underpins the G2/M cell cycle arrest induced by our compounds. Also, compounds 6f and 9f fundamentally resulted in entering MDA-PATC53 cells into the early stage of apoptosis with good percentages compared to the positive control Sunitinib. The in silico molecular-docking outcomes of scaffolds 6a-o and 9a-p in VEGFR-2, PDGFRα, and PDGFRβ active sites depicted their ability to adopt essential binding interactions like the reference Sunitinib. Our designed analogs, specifically 6f and 9f, possess promising antiproliferative and kinase inhibitory properties, making them potential candidates for further therapeutic development.

Kinase inhibitor pulldown assay (KiP) for clinical proteomics

Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

The frequency of mutations in advanced thyroid cancer in Japan: a single-center study.

We analyzed the outcomes of genetic testing to study the frequency of mutations in advanced thyroid cancer in Japan. Patients (n = 96) with unresectable or metastatic thyroid carcinoma were included for retrospective chart review. Results of gene panel testing, which was performed between May 2020 and April 2023, were analyzed. The median age of the patients was 73.5 years (range, 17-88); 59 were women, and 39 were men. Overall, 17 patients had anaplastic thyroid carcinoma (ATC), 68 had papillary thyroid carcinoma (PTC), 7 had follicular thyroid carcinoma, and 6 had poorly differentiated thyroid carcinoma (PDTC). Of the 81 patients with differentiated thyroid carcinoma (DTC) and PDTC, 88.9% were radioactive iodine-refractory, and 32.7% of all cases had previously been treated with multiple kinase inhibitors. Of ATC cases, 52.9% had BRAF mutations, and 5.9% had RET fusion. Of PTC cases, 83.1% had BRAF mutations, 9.2% had RET fusion, and 1.5% had NTRK fusion. One case each of ATC and PTC had a tumor mutation burden of ≥10. ATC cases had a significantly higher prevalence of TP53 alterations than the other cases (82.3% vs. 11.8%), whereas the frequencies of TERT promoter mutations were 88.2% in ATC cases and 64.7% in the other cases, albeit without a significant difference. In conclusion, 58.8% of ATC, 93.8% of PTC, and 42.9% of PDTC had genetic alterations linked to therapeutic agents. Active gene panel testing is required to increase treatment options.

Sorafenib combination with imatinib enhances apoptotic and cell cycle arrest effects with no effect on viability and BCR-ABL expression of K-562 cells

Chronic myeloid leukemia (CML) is one of the myeloproliferative disorders that affect hematopoiesis. Historically, many therapies have been used for the management of this disease with moderate response. The discovery of tyrosine kinase inhibitors (TKIs), marked by the introduction of imatinib, revolutionized the therapy of CML. Resistance to imatinib, however, marks CML cells in refractory disease. Additionally, advanced stages of CML may still be refractory to available therapies. Sorafenib is an inhibitor of multiple intracellular and receptor protein kinases that is approved by the FDA for the treatment of advanced hepatocellular carcinoma patients and renal cell cancer. Other TKIs, such as erlotinib and sunitinib, are under study for the treatment of CML patients. In this study, the synergistic effect of the combination of imatinib and sorafenib on the K-562 CML cell line was evaluated. We used viability assay, cell cycle analysis, apoptosis assay, reverse transcription quantitative real-time PCR detect the expression of Breakpoint Cluster Region – abelson fusion oncogene and to evaluate the interaction of the combination. The study showed that the addition of sorafenib to imatinib did not produce a significant synergistic effect on the viability of cells. The combination, however, had a significant effect on cell cycle arrest and induction of apoptosis with no effect on the reduction of BCR-ABL mRNA expression on K-562 cells.