Multi-tyrosine Kinase Inhibitor Research Articles

Lenvatinib halts aortic aneurysm growth by restoring smooth muscle cell contractility.

Abdominal aortic aneurysm (AAA) is a disease with high morbidity and mortality, especially when ruptured. The rationale of this study was to evaluate the repurposing of lenvatinib, a multi–tyrosine kinase inhibitor, in limiting experimental AAA growth targeting vascular smooth muscle cells (VSMCs) and angiogenesis. We applied systemic and local lenvatinib treatment to elastase-induced murine aortic aneurysms, and RNA profiling identified myosin heavy chain 11 (Myh11) as the most deregulated transcript. Daily oral treatment substantially reduced aneurysm formation in 2 independent mouse models. In addition, a large animal aneurysm model in hypercholesterolemic low-density lipoprotein receptor–knockout (LDLR–/–) Yucatan minipigs was applied to endovascularly deliver lenvatinib via drug-eluting balloons (DEBs). Here, a single local endovascular delivery blocked AAA progression successfully compared with a DEB-delivered control treatment. Reduced VSMC proliferation and a restored contractile phenotype were observed in animal tissues (murine and porcine), as well as AAA patient-derived cells. Apart from increasing MYH11 levels, lenvatinib reduced downstream ERK signaling. Hence, lenvatinib is a promising therapy to limit aortic aneurysm expansion upon local endovascular delivery. The tyrosine kinase inhibitor was able to positively affect pathways of key relevance to human AAA disease, even in a potentially new local delivery using DEBs.

Dual Targeting of Sorafenib-Resistant HCC-Derived Cancer Stem Cells.

Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.

Connexin 43 and Connexin 26 Involvement in the Ponatinib-Induced Cardiomyopathy: Sex-Related Differences in a Murine Model.

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.

Phase I trial of fourth-generation chimeric antigen receptor T-cells targeting glypican-3 for advanced hepatocellular carcinoma.

4088 Background: GPC3 is highly expressed in hepatocellular carcinoma (HCC) and is a promising target for HCC immunotherapy. Earlier phase I trial results demonstrated that second-generation GPC3-specific chimeric antigen receptor (CAR-GPC3) T cells were well tolerated in advanced HCC patients and showed clinical benefits (Shi D, Clin Can Res, 2020). Further preclinical studies showed that CAR-GPC3 T cells combined with multi-tyrosine kinase inhibitors (TKIs) were more effective in killing GPC3+ HCC xenografts (Wu X, Mol Ther, 2019), and fourth-generation (4G) CAR-GPC3 T cells co-expressing a transcription factor more effectively killed GPC3+ HCC xenografts. Methods: In this single-arm, open-label, first-in-human phase I trial (NCT03980288), we investigated the safety and antitumor activity of autologous 4G-CAR-GPC3 T cells for GPC3+ heavily pretreated advanced HCC patients. Patients received 4G-CAR-GPC3 T cell infusion(s) after lymphodepletion pretreatment and 67% patients combined with TKIs. Adverse events (AEs) were graded by CTCAE 5.0. Cytokine release syndrome (CRS) was graded by American Society for Transplantation and Cellular Therapy criteria (2019). Tumor response was assessed by RECISTv1.1. Results: As of January 30th, 2021, 6 subjects with HBV-related metastatic HCC were enrolled. All had progressed on ≥ 2 lines of systemic therapy, with at least one TKI combined with anti-PD-1/PD-L1 immunotherapy or FOLFOX4 chemotherapy. After lymphodepletion, patients were treated with 1-2 cycles (totaling 2.5–5×108 CAR+ cells) of 4G-CAR-GPC3 T cell therapy. Among them, 1 patient received half dose of sorafenib and 3 patients received half dose of regorafenib along with cell infusion. No dose limiting toxicity (DLT) occurred. The maximum tolerated dose (MTD) was not observed. No patient withdrew from the trial due to AE. No treatment-related death or neurotoxicity occurred. The most common ≥ grade 3 AEs were hematological toxicity, mainly due to lymphodepletion, and patients recovered within 2 weeks after therapy. All patients developed CRS including 3 at grade 2 and 3 at grade 3. Patients recovered from CRS after tocilizumab therapy with corticosteroid (4 patients) or without. One patient achieved partial response and continued after 18 weeks. The objective response rate (ORR) and disease control rate (DCR) were 16.7% and 50%, respectively. The median progression free survival (mPFS) was 4.2 months. The median peak CAR-GPC3 copies were 5067 copies/ug gDNA, and CAR-GPC3 copies were detectable on day 28 ranging 113–2071 copies/ug gDNA. Conclusions: Our study is the first to report that 4G-CAR-GPC3 T cell therapy in combination with TKIs has a manageable safety profile while demonstrating potential antitumor activity for heavily pretreated advanced HCC patients; however, CRS should be diligently managed. Clinical trial information: NCT03980288.

Modeling clonal structure over narrow time frames via circulating tumor DNA in metastatic breast cancer

BackgroundCirculating tumor DNA (ctDNA) offers minimally invasive means to repeatedly interrogate tumor genomes, providing opportunities to monitor clonal dynamics induced by metastasis and therapeutic selective pressures. In metastatic cancers, ctDNA profiling allows for simultaneous analysis of both local and distant sites of recurrence. Despite the promise of ctDNA sampling, its utility in real-time genetic monitoring remains largely unexplored.MethodsIn this exploratory analysis, we characterize high-frequency ctDNA sample series collected over narrow time frames from seven patients with metastatic triple-negative breast cancer, each undergoing treatment with Cabozantinib, a multi-tyrosine kinase inhibitor (NCT01738438, https://clinicaltrials.gov/ct2/show/NCT01738438). Applying orthogonal whole exome sequencing, ultra-low pass whole genome sequencing, and 396-gene targeted panel sequencing, we analyzed 42 plasma-derived ctDNA libraries, representing 4–8 samples per patient with 6–42 days between samples. Integrating tumor fraction, copy number, and somatic variant information, we model tumor clonal dynamics, predict neoantigens, and evaluate consistency of genomic information from orthogonal assays.ResultsWe measured considerable variation in ctDNA tumor faction in each patient, often conflicting with RECIST imaging response metrics. In orthogonal sequencing, we found high concordance between targeted panel and whole exome sequencing in both variant detection and variant allele frequency estimation (specificity = 95.5%, VAF correlation, r = 0.949), Copy number remained generally stable, despite resolution limitations posed by low tumor fraction. Through modeling, we inferred and tracked distinct clonal populations specific to each patient and built phylogenetic trees revealing alterations in hallmark breast cancer drivers, including TP53, PIK3CA, CDK4, and PTEN. Our modeling revealed varied responses to therapy, with some individuals displaying stable clonal profiles, while others showed signs of substantial expansion or reduction in prevalence, with characteristic alterations of varied literature annotation in relation to the study drug. Finally, we predicted and tracked neoantigen-producing alterations across time, exposing translationally relevant detection patterns.ConclusionsDespite technical challenges arising from low tumor content, metastatic ctDNA monitoring can aid our understanding of response and progression, while minimizing patient risk and discomfort. In this study, we demonstrate the potential for high-frequency monitoring of evolving genomic features, providing an important step toward scalable, translational genomics for clinical decision making.

Advanced Adrenocortical Carcinoma: Current Perspectives on Medical Treatment.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy. For stage I and II tumors, surgery is a curative option, but even in these cases recurrence is frequent. Practical guidelines advocate a combination of mitotane with etoposide, doxorubicin, and cisplatin as first-line therapy for metastatic adrenocortical carcinoma. However, this scheme presents limited efficacy and high toxicity. The use of Immune Checkpoint Inhibitors (ICI) and multi-Tyrosine Kinase Inhibitors (mTKI) has modified the approach of multiple malignancies. The expectation of their applicability on advanced adrenocortical carcinoma is high but the role of these new therapies persists unclear. This article provides a short summary of last years' findings targeting outcomes, limitations, and adverse effects of these new therapeutic approaches. The results of recent trials and case series pointed pembrolizumab as the most promising drug among these new therapies. It is the most often used ICI and the one presenting the best results with less related adverse effects when in comparison to the standard treatment with mitotane. Hereafter, the identification of specific molecular biomarkers or immune profiles associated with ICI or mTKI good response will facilitate the selection of candidates for these therapies. So far, microsatellite instability and Lynch Syndrome related germline mutations are suggested as predictive biomarkers of good response. Contrarywise, cortisol secretion has been associated with more aggressive ACC tumors and potentially poor responses to immunotherapy.

Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment.

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib.

Safety and activity of vandetanib in combination with everolimus in patients with advanced solid tumors: a phase I study

BackgroundPreclinical studies suggest that combining vandetanib (VAN), a multi-tyrosine kinase inhibitor of rearranged during transfection (RET) proto-oncogene, vascular endothelial growth factor receptor (VEGFR), and epidermal growth factor receptor (EGFR), with everolimus (EV), a mammalian target of rapamycin (mTOR) inhibitor, may improve antitumor activity. We determined the safety, maximum tolerated dose (MTD), recommended phase II dose (RP2D), and dose-limiting toxicities (DLTs) of VAN + EV in patients with advanced solid cancers and the effect of combination therapy on cancer cell proliferation and intracellular pathways.Patients and methodsPatients with refractory solid tumors were enrolled in a phase I dose-escalation trial testing VAN (100-300 mg orally daily) + EV (2.5-10 mg orally daily). Objective responses were evaluated using RECIST v1.1. RET mutant cancer cell lines were used in cell-based studies.ResultsAmong 80 patients enrolled, 72 (90%) patients were evaluable: 7 achieved partial response (PR) (10%) and 37 had stable disease (SD) (51%; duration range: 1-27 cycles). Clinical benefit (SD or PR ≥ 6 months) was observed in 26 evaluable patients [36%, 95% confidence intervals (CI) (25% to 49%)]. In 80 patients, median overall survival (OS) was 10.5 months [95% CI (8.5-16.1)] and median progression-free survival (PFS) 4.1 months [95% CI (3.4-7.3)]. Six patients (7.5%) experienced DLTs and 20 (25%) required dose modifications. VAN + EV was safe, with fatigue, rash, diarrhea, and mucositis being the most common toxicities. In cell-based studies, combination therapy was superior to monotherapy at inhibiting cancer cell proliferation and intracellular signaling.ConclusionsThe MTDs and RP2Ds of VAN + EV are 300 mg and 10 mg, respectively. VAN + EV combination is safe and active in refractory solid tumors. Further investigation is warranted in RET pathway aberrant tumors.

Anti-VEGF Mediated Immunomodulatory Role of Phytochemicals: Scientific Exposition for Plausible HCC Treatment.

Hepatocellular carcinoma (HCC) is one of the most common solid tumours and the second leading cause of cancer-related mortality worldwide. Advanced-recurrent HCC often requires a systemic drug therapy where multi tyrosine kinase inhibitor, Sorafenib represents the first-line therapy option. But it exhibited very limited survival benefit and tumour response due to the early emergence of drug resistance and drug-related adverse effect. Immunotherapy approaches now being widely studied as an effective alternative treatment for HCC. Several immune checkpoint inhibitors (ICI), such as Nivolumab and Pembrolizumab, are approved as monotherapy in sorafenib-resistant HCC patients. But, the existence of a plethora of immunosuppressive signals in the tumour microenvironment often leads to unsuccessful immunotherapies. In this context, combinatorial immunotherapies are getting much acceptance as a way to improve therapeutic outcomes by blocking immunosuppressive signals in the tumour microenvironment (TME). The combination of Vascular Endothelial Growth Factor (VEGF) inhibitors with ICI resulted in significant synergistic effects in various preclinical and clinical studies. However, the adverse effects associated with current synthetic VEGF inhibitors limit its clinical utility. In this review, we have summarized the potential of phytochemicals, especially the category of flavonoids, alkaloids, glycosides, terpenoids, and coumarin, as the available-affordable-safe-effective repositories of VEGF inhibitors. Their possibilities as an alternative for synthetic VEGF inhibitors by synergistic combination with ICI are reviewed, thereby enhancing patient compliance and survival rates. This review highlights the demand for a detailed investigation of the plausible role of plant-based anti-angiogenic-immunotherapy combination against HCC.

Durable response achieved using Pazopanib for germ tumor cells: A case report.

Germ cell tumors with somatic-type malignancy (GCTSTM) are rare, and patients with GCTSTM have extremely poor prognoses with a median survival of nine months. Somatic-type malignancy, which are mainly sarcomas, usually exhibit chemoresistance. The recommended therapy for GCTSTM is radical resection; however, Pazopanib, which is a multityrosine kinase inhibitor, has indicated therapeutic effects for some soft tissue sarcoma components. The current study reports the case of a 21-year-old Asian man who presented with GCTSTM after combined chemotherapy for a primary mediastinal germ cell tumor with multiple lung metastases. Despite the metastases, his disease was stable after continuous administration of Pazopanib for two years and then stopping the medication for four years. To the best of our knowledge, the current report is the first report of a durable response by Pazopanib for GCTSTM, which is a rare outcome.

Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. Objectives: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA2020 and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. Methods: The PamChip® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. Results: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA2020 with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA2020 with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG1/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G1 phase, p21/p27, and apoptosis in HT29-5FU cells. Conclusion: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA2020 and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.

Traditional Chinese Medicine Brucea Javanica Oil Enhances the Efficacy of Anlotinib in a Mouse Model of Liver-Metastasis of Small-cell Lung Cancer

Small-cell lung cancer (SCLC) is a recalcitrant disease with liver and other metastasis. The present study evaluated the efficacy of the traditional Chinese medicine Brucea javanica oil (BJO) combined with anlotinib, a multi-tyrosine kinase inhibitor with anti-angiogenic activity, on a nude-mouse model of SCLC liver metastasis. The mouse model was established by injecting NCI-H446 cells (1×106) in Matrigel (20 μl) into the upper liver lobe. All animals were randomized and assigned to three groups: Control (n=8); anlotinib alone (n=8; 3 mg/kg, qd×14+7-day interval with two cycles, oral); anlotinib plus BJO (n=8; 3 mg/kg anlotinib qd×14+7-day interval with two cycles, orally; BJO: 1 g/kg, qd×6 weeks, orally). Body weight was determined every week. Six weeks after initial treatment, tumors were collected for analysis of angiogenesis using immunohistochemistry. The combination of anlotinib and BJO significantly inhibited growth of SCLC liver metastases and angiogenesis more than anlotinib monotherapy (p=0.043). In addition, BJO alleviated body-weight loss associated with anlotinib therapy, including general mouse condition. The results of the present study indicate that the combination of anlotinib with BJO is promisingly active against liver metastases of SCLC, and has clinical potential.

Immunotherapy and chimeric antigen receptor T-cell therapy in hepatocellular carcinoma

Advanced hepatocellular carcinoma (HCC) is a deadly disease. With increasing incidence of new cases over the last years multiple efforts have been made to ameliorate survival and quality of life. Recent advances in understanding the tumor microenvironment and cancer immune evasion led to development of potent immune therapies targeting programmed death-ligand-1 (PD-L1), programmed cell death protein 1 (PD-1) and anti-cytotoxic T-lymphocyte-associated protein-4 (CTLA-4). Early clinical studies highlighted the activity and synergism of checkpoint inhibitors with antiangiogenic drugs, including anti-vascular endothelial growth factor (VEGF) antibodies and multi-tyrosine kinase inhibitors. Most recently, the combination of bevacizumab and atezolizumab improved survival compared to sorafenib in the treatment of advanced HCC on first-line therapy in a randomized phase III trial and now is considered the standard of care. Multiple options are available for the treatment of metastatic HCC including atezolizumab, bevacizumab, nivolumab, pembrolizumab, ipilimumab, sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab. Furthermore, other checkpoint inhibitors are being evaluated including durvalumab, tremelimumab, CS1003, sintilimab and camrelizumab. In this review article, we focus on the landscape of different immunotherapy strategies in the management of HCC and the combination of checkpoint inhibitors antibodies with antiangiogenics. In addition, we will address the limitation of cell therapies in advanced HCC and current strategies to improve efficacy.

Hepatic Stem Cell-like Subtypes of Hepatocellular Carcinoma Revealed from the Integrative Multi-omics Analysis Using Developmental Hierarchies of the Liver

Background: Hepatocellular carcinoma (HCC) is lethal malignancy showing high relapse rates after curative resection in early-stage. Aggressive tumor biology in resectable HCC remains unclear. Methods: Using human fetal liver signatures, multi-omics dataset from multiple clinical HCC cohorts were analyzed comprehensively to reveal molecular mechanisms for HCC stemness as well as potential biomarkers to enhance therapeutic efficacy for molecular targeted therapy or immunotherapy in stem cell-like HCC subtypes. Results: The patients predicted to the hepatic stem cell (HS) subtype showed aggressive tumor features including large tumor size, high AFP, vascular invasion, and extrahepatic metastasis as well as worst prognosis with early recurrence even in early-stage. The oncogenic pathways in terms of cell cycle, epithelial-mesenchymal transition, and TGF-beta pathway were highly upregulated in the HS subtype. Higher mutations of TP53, RB1 with PTEN deletion were significantly identified in the HS subtype. We also identified subtype-specific tissue and serum biomarkers. Predicted responders for immunotherapy were significantly lower in stem cell-like subtypes due to higher accumulation of TAM and MDSC. The HS subtype showed potential higher response to multi-tyrosine kinase inhibitors, especially sorafenib and lenvatinib. Conclusion: Stem cell-like HCC is not only associated with significantly higher relapse rate after curative resection but also with molecular biology for the aggressive subtype of HCC. We identified subtype-specific serum and tissue biomarkers for stem cell-like subtypes and precise therapeutic strategies for each subtype regarding immunotherapy and molecular-targeted treatment. Our findings may offer theoretical foundation of biomarker-based clinical trials for new therapeutic approaches to resectable early-stage HCC patients.

Programmed Death Ligand-1 (PD-L1) Regulated by NRF-2/MicroRNA-1 Regulatory Axis Enhances Drug Resistance and Promotes Tumorigenic Properties in Sorafenib-Resistant Hepatoma Cells.

Sorafenib, a multityrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. Programmed death ligand-1 (PD-L1) is one of the most important inhibitory molecules involved in tumor immune evasion. Recently, it has been reported that PD-L1 could play crucial roles in drug resistance of many kinds of cancers. However, the expression, function, and regulation of PD-L1 in sorafenib-resistant hepatoma cells remain unclear. In this study, we reported that PD-L1 was overexpressed in sorafenib-resistant hepatoma cells, and shRNA-mediated PD-L1 depletion attenuated drug resistance and suppressed the migration, invasion, colony formation, and tumorigenesis in sorafenib-resistant hepatoma cells in vitro and in vivo. Mechanistic investigations indicated that loss of microRNA-1 (miR-1), a tumor-suppressive microRNA, contributed to the PD-L1 upregulation in sorafenib-resistant hepatoma cells, and PD-L1 was a direct regulatory target of miR-1. Further study revealed that an oncogenic transcriptional factor, nuclear factor E2-related factor 2 (NRF-2), was induced in sorafenib-resistant hepatoma cells and inhibited expression of miR-1 in vitro. From molecular mechanism insight back to the functional verification, we eventually demonstrated that miR-1 executed its tumor-suppressive effects on drug resistance and other malignant properties in sorafenib-resistant hepatoma cells partially by PD-L1 inhibition in vitro and in vivo. In conclusion, our data suggested that a NRF-2/miR-1/PD-L1 regulatory axis contributed to the development and maintenance of drug resistance and other tumorigenic properties in sorafenib-resistant hepatoma cells and provided a potential therapeutic target for overcoming sorafenib resistance in HCC.

Reproducible and Characterized Method for Ponatinib Encapsulation into Biomimetic Lipid Nanoparticles as a Platform for Multi-Tyrosine Kinase-Targeted Therapy.

Ponatinib (Pon) is a multi-tyrosine kinase inhibitor that demonstrated high efficiency for treating cancer. However, severe side effects caused by Pon off-targeting effects prevent its extensive use. Using our understanding into the mechanisms by which Pon is transported by bovine serum albumin in the blood, we have successfully encapsulated Pon into a biomimetic nanoparticle (NP). This lipid NP (i.e., "leukosomes") incorporates membrane proteins purified from activated leukocytes that enable immune evasion, and enhanced targeting of inflamed endothelium NPs have been characterized for their size, charge, and encapsulation efficiency. Membrane proteins enriched on the NP surface enabled modulation of Pon release. These NP formulations showed promising dose-response results on two different murine osteosarcoma cell lines, F420 and RF379. Our results indicate that our fabrication method is reproducible, nonuser-dependent, efficient in loading Pon, and applicable toward repurposing numerous therapeutic agents previously shelved due to toxicity profiles.

Immune Checkpoint Inhibitors versus VEGF Targeted Therapy as Second Line Regimen in Advanced Hepatocellular Carcinoma (HCC): A Retrospective Study.

Several targeted agents including multi-tyrosine kinase inhibitors (mTKIs) and immunotherapy (IO) agents have been approved for use beyond the frontline setting in patients with advanced hepatocellular carcinoma (HCC). Due to lack of prospective head-to-head comparative trials, there is no standardized way for alternating those agents beyond frontline. Therefore, we performed a retrospective review of the Kansas University (KU) cancer registry to determine whether IO may be superior to non-IO therapy. Patients with advanced HCC were divided into two groups based on the second-line systemic regimen received (IO vs. non-IO). Progression-free survival (PFS) and overall survival (OS) were calculated under the Kaplan–Meier and Cox proportional hazards models. No statistically significant differences in PFS and OS were found, although a non-significant delayed separation in the survival curve favoring IO was identified (median PFS 3.9 months vs. 3 months; median OS 10 months vs. 10 months respectively for IO vs. non-IO). This retrospective analysis is one of the earliest and largest studies comparing second-line IO and non-IO therapies thus far reported. Future studies should aim to define specific biomarkers for response prediction and treatment optimization based on individual patient and tumor characteristics. Furthermore, combinatorial therapeutic strategies is an evolving approach showing early promising signal.

Abstract 5720: MicroRNA deregulation of the serine synthesis pathway controls intrinsic and non-cell autonomous mechanism of resistance to Regorafenib in metastatic colorectal cancer (mCRC)

Abstract Background: MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis. miR dysregulation has been linked with activation of oncogenic pathways, cancer progression and clinical outcome in mCRC. Chemo-refractory mCRC patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient stratification and identification of mechanisms of resistance. Methods: Serial liquid biopsies were obtained at baseline (BL) and monthly until disease progression (PD) in 43 patients treated with regorafenib for chemo-refractory mCRC in the context of a phase II clinical trial (PROSPECT-R). Tissue biopsies were obtained at BL, after 2 months and at PD within the same trial and used to establish Patient-Derived Organoids (PDOs) and for molecular analyses. PDOs co-cultures and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. Liquid biopsies were also obtained from an additional cohort (n=97) of mCRC patients treated with regorafenib. MiR profiling was performed on baseline seras using NanoString nCounter platform and significant miRs were validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Functional experiments were performed in PDOs, PDO co-cultures and PDO-xenotransplants. Results: MiR expression was tested in 43 BL in the PROSPECT-R trial. Up-regulation of miR-652-3p was associated with poor PFS and OS. These results were validated by ddPCR on the same serum samples, matching plasmas and organoids. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. The same findings were confirmed in the validation cohort. Functional experiments showed that miR-652-3p upregulation has significant effects on cancer cell migration. Up and down-regulation of miR-652-3p upon regorafenib treatment translated in a significant effect on cell viability in PDO co-cultures and liver PDO-xenotransplants. RNA-sequencing analysis of miR-652-3p over-expressing organoids showed downregulation of several components of the serine synthesis pathway. Among them, phosphoserine aminotransferase (PSAT1) was validated as a miR-652-3p direct target. Rescue experiments confirmed that PSAT1 over-expression and silencing lead to increase sensitivity and resistance to regorafenib respectively via cell and non-cell autonomous regulation of autophagy. Conclusions: Our data suggest that miR-652-3p may be uses as a prognostic/predictive biomarker for the selection of treatment and provide mechanics insight on regorafenib resistance. Citation Format: Somaieh Hedayat, Andrea Lampis, George Vlachogiannis, khurum Khan, Silvia Marchetti, Matteo Fassan, Michele Ghidini, Ruwaida Begum, Marta Schirripa, Rodolfo Passalacqua, David Cunningham, Fotios Loupakis, Nicola Valeri. MicroRNA deregulation of the serine synthesis pathway controls intrinsic and non-cell autonomous mechanism of resistance to Regorafenib in metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5720.

Abstract 5610: The multi tyrosine kinase inhibitor TAS-115 promotes innate and adaptive immune responses of androgen deprivation therapy in mouse prostate cancer

Abstract We previously used a transgenic mouse model of prostate cancer to show that TAS-115, a multi-kinase inhibitor that targets c-MET, VEGF-R, and CSF1-R signal pathways, effectively reduced growth in a subset of prostate tumors. In this setting, TAS-115 decreased tumor burden by reducing tumor neovascularization and promoting anti-tumor immune activity, however, this phenomenon was not observed when co-administered with androgen deprivation therapy (ADT) which is the primary treatment option for men with advanced prostate cancer. We have also shown that androgen withdrawal can promote T cell infiltration, however, antitumor immune responses were hampered most likely by the increased prevalence of immune suppressive cells consisting of tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) induced after ADT. Our previous findings revealed that TAS-115 was able to suppress M2 polarized macrophages and promote the recruitment and activity of tumoricidal neutrophils in castration-naïve tumors. We surmised that pretreatment with TAS-115 could potentiate antitumor immunity of ADT-induced inflammation by decreasing immune suppressive cell activity. In this follow-up study, we demonstrate that one-week treatment with TAS-115 modified the composition of immune cells in intact prostate tumors from Pten/Trp53-deficient conditional knockout mice. Immunophenotyping studies showed that mice treated with TAS-115 had reduced levels of tumor and circulating monocytic-MDSCs (m-MDSC, CD11b+/Ly6C+) and TAMs. Expression levels of genes associated with m-MDSCs and M2 polarized macrophages (Arg1, Csf1r, Il10, Nos2, Adgre1, CD163, Cd68 and Tnf) were also decreased in TAS-115-treated mice. Increases of total and CD355+(CRTAM) CD8 T cells indicating enhanced recruitment and activation were observed in both tumors and blood of TAS-115 treated mice. Sequencing TAS-115 with or without surgical castration was evaluated in transgenic mice bearing Pten/Trp53-deificent prostate tumors. Mice treated for four weeks with TAS-115/ADT sequential therapy showed a 13.5% (P=0.0836) reduction of tumor burden compared to vehicle-treated controls. Notably, mice treated with TAS-115/ADT sequential therapy were characterized with a decrease of monocytic and granulocytic MDSCs in tumors. Moreover, neutrophil mediated antitumor activity was increased in TAS-115 treated mice. We also use phenotyping, gene expression and immunohistochemical studies to show that reducing immunosuppressive immune cells led to increased CD8 T cell recruitment and activation. These findings provide evidence to support that TAS-115 sequenced with ADT has the potential to augment innate and adaptive antitumor immunity in prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. The multi tyrosine kinase inhibitor TAS-115 promotes innate and adaptive immune responses of androgen deprivation therapy in mouse prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5610.

Abstract 2202: The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity

Abstract ALKS 4230, an engineered cytokine designed for selective binding to the intermediate affinity interleukin-2 receptor (IL-2R), is being evaluated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. The selectivity of ALKS 4230 is achieved through stable fusion of circularly permuted IL-2 to IL-2Rα, with the goal of driving antitumor responses via selective activation of CD8+ T cells and NK cells, while avoiding activation of regulatory T cells (Tregs), which express the high affinity IL-2R. High levels of vascular endothelial growth factor (VEGF) are associated with an immunosuppressive tumor microenvironment and diminished response to high-dose IL-2. Lucitanib is an anti-angiogenic, multi-tyrosine kinase inhibitor targeting VEGFR1-3, PDGFRα/β, and FGFR1-3. We evaluated the antitumor efficacy and mechanism of the combination of RDB 1462, the mouse ortholog of ALKS 4230, and lucitanib in the MC38 mouse syngeneic tumor model. C57BL/6 mice were subcutaneously implanted with MC38 cells and treated with RDB 1462 (1.5 mg/kg and 3 mg/kg once every 3 days for 3 weeks), lucitanib (10 mg/kg daily for 28 days), or the combination of both molecules. After 11 days of treatment, lucitanib monotherapy resulted in 100% tumor growth inhibition (TGI), while 1.5 mg/kg and 3 mg/kg RDB 1462 monotherapy resulted in 25% and 51% TGI, respectively. Lucitanib monotherapy extended survival, with a mean survival time (MST) of 39 days compared to 15 days for vehicle, with no complete tumor regressions. MSTs were 18-22 days for RDB 1462 monotherapy groups, 60 days for the lower dose combination arm, and >60 days for the higher dose combination. Durable complete tumor regressions were observed in 1/20 mice treated with either dose of RDB 1462 monotherapy and 5/10 mice treated with the lower dose of RDB 1462 in combination with lucitanib. 10/10 mice treated with the higher dose of RDB 1462 in combination with lucitanib exhibited complete responses on Day 60. Flow cytometric analyses revealed that RDB 1462 treatment induced significant peripheral CD8+ T cell expansion compared to vehicle (p<0.05) without an increase in Foxp3+ Tregs. This effect was enhanced by the combination of RDB 1462 with lucitanib. The combination led to a significant (p<0.05) increase in intratumoral CD8+ T cells and dendritic cells (CD11c+F4/80−), together with a reduction in tumor-associated macrophages (CD11b+F4/80+). The combination treatment elicited a distinct gene expression profile in tumors by RNA-Seq, merging the immunostimulatory effects of RDB 1462 with the anti-angiogenic effects of lucitanib. The combination of an intermediate affinity IL-2R-selective cytokine and an angiogenesis inhibitor resulted in durable dose-dependent antitumor efficacy in the MC38 mouse tumor model. Citation Format: Jared E. Lopes, Rachel L. Dusek, Liliane Robillard, Minh Nguyen, Bruce Roth, Bryan Vought, Arthur Liberzon, Raymond J. Winquist, Heather C. Losey. The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2202.