Sorafenib Exposure Research Articles

Sodium-Glucose Co-Transporter-2 Inhibitor Empagliflozin Attenuates Sorafenib-Induced Myocardial Inflammation and Toxicity.

Environmental antineoplastics such as sorafenib may pose a risk to humans through water recycling, and the increased risk of cardiotoxicity is a clinical issue in sorafenib users. Thus, developing strategies to prevent sorafenib cardiotoxicity is an urgent work. Empagliflozin, as a sodium-glucose co-transporter-2 (SGLT2) inhibitor for type 2 diabetes control, has been approved for heart failure therapy. Still, its cardioprotective effect in the experimental model of sorafenib cardiotoxicity has not yet been reported. Real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to study the effect of sorafenib exposure on cardiac SGLT2 expression. The impact of empagliflozin on cell viability was investigated in the sorafenib-treated cardiomyocytes using Alamar blue assay. Immunoblot analysis was employed to delineate the effect of sorafenib and empagliflozin on ferroptosis/proinflammatory signaling in cardiomyocytes. Ferroptosis/DNA damage/fibrosis/inflammation of myocardial tissues was studied in mice with a 28-day sorafenib ± empagliflozin treatment using histological analyses. Sorafenib exposure significantly promoted SGLT2 upregulation in cardiomyocytes and mouse hearts. Empagliflozin treatment significantly attenuated the sorafenib-induced cytotoxicity/DNA damage/fibrosis in cardiomyocytes and mouse hearts. Moreover, GPX4/xCT-dependent ferroptosis as an inducer for releasing high mobility group box 1 (HMGB1) was also blocked by empagliflozin administration in the sorafenib-treated cardiomyocytes and myocardial tissues. Furthermore, empagliflozin treatment significantly inhibited the sorafenib-promoted NFκB/HMGB1 axis in cardiomyocytes and myocardial tissues, and sorafenib-stimulated proinflammatory signaling (TNF-α/IL-1β/IL-6) was repressed by empagliflozin administration. Finally, empagliflozin treatment significantly attenuated the sorafenib-promoted macrophage recruitments in mouse hearts. In conclusion, empagliflozin may act as a cardioprotective agent for humans under sorafenib exposure by modulating ferroptosis/DNA damage/fibrosis/inflammation. However, further clinical evidence is required to support this preclinical finding.

A comprehensive review of the relationship between autophagy and sorafenib-resistance in hepatocellular carcinoma: ferroptosis is noteworthy.

Patients with hepatocellular carcinoma (HCC) bear a heavy burden of disease and economic burden but have fewer treatment options. Sorafenib, a multi-kinase inhibitor, is the only approved drug that can be used to limit the progression of inoperable or distant metastatic HCC. However, enhanced autophagy and other molecular mechanisms after sorafenib exposure further induce drug resistance in HCC patients. Sorafenib-associated autophagy also generates a series of biomarkers, which may represent that autophagy is a critical section of sorafenib-resistance in HCC. Furthermore, many classic signaling pathways have been found to be involved in sorafenib-associated autophagy, including the HIF/mTOR signaling pathway, endoplasmic reticulum stress, and sphingolipid signaling, among others. In turn, autophagy also provokes autophagic activity in components of the tumor microenvironment, including tumor cells and stem cells, further impacting sorafenib-resistance in HCC through a special autophagic cell death process called ferroptosis. In this review, we summarized the latest research progress and molecular mechanisms of sorafenib-resistance-associated autophagy in detail, providing new insights and ideas for unraveling the dilemma of sorafenib-resistance in HCC.

Thyroid hormone T3 augments the cytotoxicity of sorafenib in Huh7 hepatocellular carcinoma cells by suppressing AKT expression.

Hepatocellular carcinoma (HCC) is a primary cancer that poorly responds to treatment. Molecular cancer studies led to the development of kinase inhibitors, among which sorafenib stands out as a multi-kinase inhibitor approved by FDA for first line use in HCC patients. However, the efficiency of sorafenib was shown to be counteracted by numerous subcellular pathways involving the effector kinase AKT, causing resistance and limiting its survival benefit. On the way of breaking such resistance mechanisms and increase the efficiency of sorafenib, deeper understanding of hepatocellular physiology is essential. Thyroid hormones were shown to be metabolized in liver and inevitably affect the molecular behaviour of hepatocytes. Interestingly, thyroid hormone T3 was also demonstrated to be potentially influential in liver regeneration and treatment with this hormone reportedly led to a decrease in HCC tumor growths. In this study, we aimed to uncover the impact of T3 hormone on the cytotoxic response to sorafenib in HCC in vitro. We pre-treated the HCC cell line Huh-7 with T3 prior to sorafenib exposure both in 2D and 3D culture. We checked cell viability with MTT assay in 2D culture and measured the sizes of 3D spheroids with bright-field microscopy followed by a surface analysis with ImageJ. We also performed scratch assay to measure cell migration as well as western blot and qPCR to uncover affected pathways. We observed an additive effect to sorafenib's cytotoxicity both in 2D and 3D culture. Cell migration assay also confirmed our finding and pointed out a benefit of T3 hormone in HCC cell migration. Western blot experiments showed that T3 exerts its additive effect by suppressing AKT expression upon sorafenib treatment both at protein and gene expression levels. Our results open a promising new avenue in increasing sorafenib's cytotoxicity where thyroid hormone T3 is utilized to modulate AKT expression to combat resistance, and warrant further studies in the field.

Regorafenib versus Cabozantinib as a Second-Line Treatment for Advanced Hepatocellular Carcinoma: An Anchored Matching-Adjusted Indirect Comparison of Efficacy and Safety

Introduction: The tyrosine kinase inhibitors regorafenib and cabozantinib remain the mainstay in second-line treatment of advanced hepatocellular carcinoma (HCC). There is currently no clear evidence of superiority in efficacy or safety to guide choice between the two treatments. Methods: We conducted an anchored matching-adjusted indirect comparison using individual patient data from the RESORCE trial of regorafenib and published aggregate data from the CELESTIAL trial of cabozantinib. Second-line HCC patients with prior sorafenib exposure of ≥3 months were included in the analyses. Hazard ratios (HRs) and restricted mean survival time (RMST) were estimated to quantify differences in overall survival (OS) and progression-free survival (PFS). Safety outcomes compared were rates of grade 3 or 4 adverse events (AEs), occurring in >10% of patients, and discontinuation or dose reduction due to treatment-related AEs. Results: After matching adjustment for differences in baseline patient characteristics, regorafenib showed a favorable OS (HR, 0.80; 95% CI: 0.54, 1.20) and ∼3-month-longer RMST over cabozantinib (RMST difference, 2.76 months; 95% CI: −1.03, 6.54), although not statistically significant. For PFS, there was no numerical difference in HR (HR, 1.00; 95% CI: 0.68, 1.49) and no clinically meaningful difference based on RMST analyses (RMST difference, −0.59 months; 95% CI: −1.83, 0.65). Regorafenib showed a significantly lower incidence of discontinuation (risk difference, −9.2%; 95% CI: −17.7%, −0.6%) and dose reductions (−15.2%; 95% CI: −29.0%, −1.5%) due to treatment-related AEs (any grade). Regorafenib was also associated with a lower incidence (not statistically significant) of grade 3 or 4 diarrhea (risk difference, −7.1%; 95% CI: −14.7%, 0.4%) and fatigue (−6.3%; 95% CI: −14.6%, 2.0%). Conclusion: This indirect treatment comparison suggests, relative to cabozantinib, that regorafenib could be associated with favorable OS (not statistically significant), lower rates of dose reductions and discontinuation due to treatment-related AEs, and lower rates of severe diarrhea and fatigue.

The influence of sunitinib and sorafenib, two tyrosine kinase inhibitors, on development and thyroid system in zebrafish larvae

Recently, the potential toxic effects of various pharmaceuticals on the thyroid endocrine system have raised considerable concerns. In this study, we evaluated the adverse effects of sorafenib and sunitinib, two widely used anti-tumor drugs, on the developmental toxicities and thyroid endocrine disruption by using zebrafish (Danio rerio) model. Zebrafish embryos/larvae were exposed to different contentions (0, 10, 50 and 100 nM) of sorafenib and sunitinib for 96 hpf. The results revealed that waterborne exposure to sorafenib and sunitinib exhibited remarkable toxic effects on the survival and development in zebrafish embryos/larvae, which was accompanied by obvious disturbances of thyroid endocrine system (e.g., decreased T3 and T4 content, increased TSH content) and genes’ transcription changes within the hypothalamus-pituitary-thyroid (HPT) axis. In addition, we verified a strikingly abnormal thyroid gland organogenesis in zebrafish larvae in response to sorafenib and sunitinib, by assessing the development of thyroid follicles using the WISH staining of tg, the Tg (tg:GFP) zebrafish transgenic line, and histopathological analysis. Taken together, our results indicated sorafenib and sunitinib exposure could induce obvious developmental toxicities and thyroid function disruption in zebrafish embryos/larvae, which might involve a regulatory mechanism, at least in part, by destroying the thyroid follicle structure, and by disturbing the balance of the HPT axis.

UDP-glucose 6-dehydrogenase lessens sorafenib sensitivity via modulating unfolded protein response

As the first-generation targeted therapy, sorafenib remains an effective single-drug treatment for advanced hepatocellular carcinoma (HCC). Unfortunately, the existence of resistance restricts the long-term benefit of patients. UDP-glucose 6-dehydrogenase (UGDH) is the key enzyme of glucuronic acid metabolism which was largely reported in mediating drug systemic elimination. In this study, we explore its critical role in regulating sorafenib sensitivity. Here we find sorafenib exposure could activate glucuronic acid metabolism, accompanied with the elevated expression of UGDH. Interference with the route by silencing UGDH could boost HCC cells sensitivity to sorafenib. Meanwhile, the analysis of HCC patients with sorafenib treatment displayed that low UGDH expression predicted superior prognosis. Further screening assay suggested that unfolded protein response (UPR) involves in UGDH silencing-mediated apoptosis. Xenograft model confirmed that combined UGDH intervention could significantly improve sorafenib efficacy. Our results reveal the impact of sorafenib exposure on glucuronic acid metabolism reprogramming and provide UGDH as a promising target to improve sorafenib efficacy.

In desmoid-type fibromatosis cells sorafenib induces ferroptosis and apoptosis, which are enhanced by autophagy inhibition

IntroductionDesmoid-type fibromatosis (DTF) is a rare, soft tissue tumour. Sorafenib, a multikinase inhibitor, has demonstrated antitumour efficacy in DTF patients. Little is known about the underlying molecular mechanisms, which are crucial to know to further optimize systemic treatments. Here we investigated the molecular effects of sorafenib exposure on DTF and stromal cells, with an emphasis on cell death mechanisms. Material and methodsDTF primary cell cultures, with known CTNNB1 status, and primary stromal cell cultures, derived from DTF tissue, were exposed to clinically relevant concentrations of sorafenib in the presence or absence of inhibitors of ferroptosis, apoptosis and autophagy. Cell viability was determined after 24 and 48 h using MTT assays. Annexin V/PI staining, lipid peroxidation analysis and immunoblotting were performed to assess apoptosis, ferroptosis and autophagy. ResultsExposure to sorafenib caused a significant, concentration- and time-dependent decrease in cell viability in all primary DTF and stromal cell cultures. Inhibitors of ferroptosis and apoptosis protected against sorafenib-mediated cytotoxicity implicating that both cell death mechanisms are activated. Annexin V/PI stainings and lipid peroxidation analyses confirmed induction of apoptosis and ferroptosis, respectively. Autophagy inhibition enhanced the cytotoxic effect of sorafenib and led to a stronger induction of apoptosis and ferroptosis. ConclusionThis study identified ferroptosis and apoptosis as mechanisms for the sorafenib induced cell death in DTF cells as well as stromal cells. Furthermore, autophagy inhibition enhanced the cytotoxic effects of sorafenib. Knowledge of the mechanisms by which sorafenib affects DTF at a cellular level may help to optimize its clinical efficacy and mitigate toxic effects.

Zinc supplementation ameliorates sorafenib-induced cognitive impairment through ROS/JNK signaling pathway.

Sorafenib, a multiple kinase inhibitor, is widely used in cancer patients. Recently, clinical studies highlighted the relationship between cognitive deficits and sorafenib exposure. Zinc abundant in the body has been reported to exert neuroprotective activities. However, the effects of zinc supplementation on sorafenib-induced cognitive impairment are still unknown. In the current study, we verified that mice challenged with sorafenib displayed characteristic features of cognitive impairment. However, zinc treatment effectively improved these changes. Histopathological staining also showed that zinc significantly alleviated hippocampal microstructural and ultrastructural damages induced by sorafenib. Meanwhile, zinc significantly reduced sorafenib-induced ROS production and neuronal cells apoptosis in vivo and vitro. Additionally, we also investigated whether zinc protected against sorafenib-induced neuronal cells apoptosis via ROS/JNK pathway through treating SH-SY5Y cells with the NAC or the specific JNK activator anisomycin. The results indicated that NAC performed the same protective effects as zinc in sorafenib-challenged SH-SY5Y cells and activation of JNK by anisomycin partly abolished the protective effects of zinc. Collectively, the present study suggested that inhibition of oxidative stress and the JNK pathway might contribute to the protective effects of zinc against sorafenib-caused cognitive impairment in vivo and vitro.

In Vitro Investigation of Renal Cell Carcinoma Response to Combination Sorafenib and Cryoablation Treatment

The 5-year survival rate for localized kidney cancer is 93%, but only 13% for those presenting with metastatic disease (2019 SEER data). Cryosurgery is an established treatment modality for renal cell cancer (RCC), with outcomes showing equipoise to radiofrequency ablation (RFA) and partial nephrectomy. Sorafenib is a targeted therapy for RCC utilized in more advanced stage diseases. Given the success of both cryoablation and sorafenib as monotherapies for RCC, in this study, we investigated the cellular response of RCC to combinatorial sorafenib pre-treatment and cryoablation in vitro using cell culture and tissue-engineered tumor models. In vitro samples were exposed to a single or repeat (double) 5-minute freeze at -10°C, -15°C, or -20°C representing temperatures within the periphery of a cryolesion. A repeat freeze to -20°C was necessary to fully ablate samples yielding day 1 viability of 2.9% (±0.2) with no recovery observed over the 7 days post-treatment culture. These findings were consistent with published data on the lethal temperature in RCC, suggesting that -25°C is necessary to destroy RCC following a single freeze event. Pre-treatment of samples with sorafenib at concentrations of 10.61 and 21.21 µM (½ clinical and clinical dose, respectively) was combined with a single or repeat 5-minute freeze to -10°C, -15°C, or -20°C. At the time of drug removal (day 0/pre-freeze), 10.61 µM sorafenib treated samples yielded 25.3% (±0.4) viability, yet samples regrew to control levels by day 7. Following combination freeze and sorafenib exposure, sample viability was found to be 27.5% (±0.7), 2.9% (±0.4), and 0.2% (±0.02) following a single freeze and 15.6% (±0.5), 0.7% (±0.1), and 0.1% (±0.01) following a repeat (double freeze), respectively. Regrowth was observed over the 7-day assessment period in samples exposed to a -10°C single or double freeze and a -15°C single freeze, but not in the -20°C single freeze or -15°C double freeze conditions. Thus, pre-treatment with 10.61 µM sorafenib was found to increase the minimum lethal temperature from the reported -25°C to -20°C following a single freeze event and from -20°C to -15°C following a double freeze. Results of the cell culture studies were confirmed in the 3D tissue-engineered tumor model, wherein the combination of 10.61 µM sorafenib and freezing was found to further increase the lethal temperature from <-20°C to -15°C following a single freeze event. This increased freeze susceptibility yielded a 32% improvement in the overall ablative volume of the ice ball following combinatorial treatment versus freezing alone. These in vitro results suggest that the combination of sorafenib and cryoablation may provide a possible combinatorial treatment path for RCC.

Pyruvate Dehydrogenase (PDH) Activity Is a Source of Resistance to Sorafenib in FLT3-ITD Expressing Acute Myeloid Leukemia (AML)

Background:AML is characterized by poor prognoses and resistance to therapy. Despite decades of research long term survival remains 30-40%, and little is known about the mechanisms involved in resistance. Cells that are resistant to cytarabine have increased oxidative metabolism and mitochondrial mass, thus suggesting a role of mitochondrial metabolism in resistance to therapy. Our lab has previously shown that genetic and pharmacologic inhibition of pyruvate dehydrogenase (PDH) sensitizes AML cells to DNA damaging agents, specifically cytarabine and doxorubicin. CPI-613 is a first in class agent that inhibits PDH and has been shown to sensitize AML cells to DNA damaging agents. How AML cells alter their mitochondrial metabolism in response to targeted therapies is not well known. Sorafenib is a tyrosine kinase inhibitor that has been shown to inhibit FLT3. Activating mutations in FLT3 are found in about 30% of AML patients with the most common mutation being an internal tandem duplication (FLT3-ITD). Sorafenib has limited activity as a single agent against FLT3 mutated AML. The degree to which mitochondrial metabolism contributes to resistance to sorafenib is currently unknown.Methods:A murine AML cell line expressing the FLT3-ITD (MFL2) was used to knockout PDH through the CRISPR-Cas9 system, and the safe-harbor ROSA26 locus was used as control. Knockout of PDH was confirmed by Western blot analysis. PDH knockout cells were treated with sorafenib at different doses for 24 and 72 hours. Viability assays were done using the EZQuant assay. Mitochondrial mass was assessed on PDH KO and ROSA26 control cells that were treated with sorafenib after 24-hours by staining the cells with Mitotracker and measuring uptake by flow cytometry. Apoptosis was assessed through annexin V and Propidium Iodide staining. Oxygen consumption was assessed using the Seahorse XF24 Extracellular Flux Analyzer. MOLM-13 (human AML cell line that expresses the FLT3-ITD) cells were treated with sorafenib with and without CPI-613 and combinatorial index values determined by the method of Chou and Talalay.Results:PDH deleted cells (PDH KO) were significantly more sensitive to sorafenib than ROSA control cells at several doses (p value <0.0001). This sensitivity is in part due to increased apoptosis, which was shown by a significant increase of Annexin V positive PDH KO cells compared to controls (p value <0.001). Interestingly, when mitochondrial oxygen consumption rates (OCR) were assessed after 24-hour sorafenib exposure, ROSA controls showed decreased OCR implying a direct effect on mitochondrial function. PDH KO cells showed low basal mitochondrial OCR that did not change with treatment of sorafenib, consistent with previous data. Mitochondrial mass was also assessed and ROSA controls showed an increase of mitochondrial mass with treatment of sorafenib at 24-hours while PDH KO cells showed no increase. CPI-613 synergized with sorafenib in MOLM-13 cells with a combinatorial index value of 0.581 (95% CI 0.4520-0.7107).Conclusion:Here we show that genetic deletion of PDH sensitizes FLT3-ITD positive cells to sorafenib. Mechanistically, sorafenib inhibits mitochondrial function as indicated by ROSA cells showing decreased OCR while increasing mitochondrial mass. PDH KO cells lack the mitochondrial reserve to counteract this affect resulting in increased cell death. A FLT3-ITD expressing cell line could be sensitized to sorafenib treatment by exposure to the TCA cycle inhibitor CPI-613. Considered in conjunction with previous findings, these results suggest that the PDH activity is an important factor in developing resistance to multiple classes of targeted and chemotherapeutic agents and suggests that CPI-613 could increase responses to targeted agents in AML. DisclosuresPardee:Rafael Pharmaceuticals: Employment, Research Funding.

Herbacetin Broadly Blocks the Activities of CYP450s by Different Inhibitory Mechanisms.

Herbacetin is a bioactive flavanol compound that has various pharmacological effects. However, the pharmacokinetic characteristics have not been thoroughly investigated. Previously, we screened a natural compound library and identified herbacetin as a potent CYP blocker. Herein, we aimed to mechanistically determine the inhibitory effects of herbacetin on CYP450 and its potential application. A human liver microsome incubation system was developed based on a UPLC-MS/MS method. Moreover, an in silico docking assay and a human CYP recombinase reaction system were developed and used to investigate binding affinity and inhibitory efficacy. Subsequently, the effects of the combination of herbacetin and sorafenib on HepG2 cells were assessed by MTT and immunoblotting assays. The concentration of sorafenib and its main metabolite were measured by UPLC-MS/MS after incubation with or without herbacetin. As a result, we found herbacetin almost completely inhibited the functions of major CYPs at 100 µM. Moreover, through analysis of the structure-activity relationship, we found 4-, 6-, and 8-hydroxyl were essential groups for the inhibitory effects. Herbacetin inhibited CYP3A4, CYP2B6, CYP2C9, and CYP2E1 in a mixed manner, but non-competitively blocked CYP2D6. These results are in good agreement with the recombinase reaction in vitro results, with an IC50 < 10 µM for each tested isoenzyme. Interestingly, the stimulatory effects of sorafenib on HepG2 cell apoptosis were significantly enhanced by combining with herbacetin, which was associated with increased sorafenib exposure. In summary, herbacetin is a potent inhibitor of a wide spectrum of CYP450s, which may enhance the exposure of drugs in vivo.

Anchored matching-adjusted indirect comparison (MAIC) of regorafenib (REG) versus cabozantinib (CAB) in advanced hepatocellular carcinoma (HCC).

e16188 Background: REG and CAB, two therapies for second-line (2L) advanced HCC, have been evaluated vs. placebo (PBO) in randomized phase III trials, RESORCE (NCT01774344) and CELESTIAL (NCT01908426), respectively. In the absence of head-to-head studies, indirect treatment comparison (ITC) is needed to compare the efficacy and safety of REG vs. CAB. Anchored MAIC is an ITC method adjusting for cross-trial heterogeneity using PBO as the common comparator or anchor. Methods: Patient-level data from RESORCE were available to match the inclusion criteria and published baseline characteristics of CELESTIAL. 2L patients with prior sorafenib exposure of &gt;3 months were included in the analyses. Overall survival (OS), progression-free survival (PFS), and Grade 3/4 treatment-emerging adverse events (TEAEs) occurring in &gt; 10% of patients were compared. Matching was conducted for the following covariates, which were either imbalanced across trials or likely treatment effect modifiers (EMs): gender, region, Eastern Cooperative Oncology Group (ECOG), prior sorafenib duration, extrahepatic spread, macrovascular invasion, hepatitis B (HBV), hepatitis C (HCV), alcohol use, and alpha-fetoprotein (AFP) ≥400 ng/mL. Anchored MAIC were used to compare the relative effect of each treatment. To account for violation of the proportional hazards assumption, in addition to hazard ratios (HRs), restricted mean survival time (RMST) was estimated to quantify differences in OS and PFS. For safety, the relative effects were expressed as risk differences (RDs) (REG – CAB). Results: After matching, the effective sample sizes of REG and its PBO arm were 214 and 82, respectively. For OS, REG showed favorable although not statistically significant HR, and 30% longer mean survival with an RMST ratio approaching statistical significance. For PFS, there was no numerical difference in HR and no clinically meaningful difference based on RMST analyses (Table). REG showed lower incidence of diarrhea [RD of -7.1% (NE, NE)] and fatigue [RD of -6.3% (-14.6, 2.0)]. Conclusions: In this anchored MAIC, REG showed favorable OS and safety profile, especially for diarrhea and fatigue. No clinically meaningful difference was observed for PFS. The analysis could be limited by reduced sample size after matching and unobserved or missing EMs that could not be adjusted.[Table: see text]

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.

Mechanistic Modelling Identifies and Addresses the Risks of Empiric Concentration-Guided Sorafenib Dosing.

The primary objective of this study is to evaluate the capacity of concentration-guided sorafenib dosing protocols to increase the proportion of patients that achieve a sorafenib maximal concentration (Cmax) within the range 4.78 to 5.78 μg/mL. A full physiologically based pharmacokinetic model was built and validated using Simcyp® (version 19.1). The model was used to simulate sorafenib exposure in 1000 Sim-Cancer subjects over 14 days. The capacity of concentration-guided sorafenib dose adjustment, with/without model-informed dose selection (MIDS), to achieve a sorafenib Cmax within the range 4.78 to 5.78 μg/mL was evaluated in 500 Sim-Cancer subjects. A multivariable linear regression model incorporating hepatic cytochrome P450 (CYP) 3A4 abundance, albumin concentration, body mass index, body surface area, sex and weight provided robust prediction of steady-state sorafenib Cmax (R2 = 0.883; p < 0.001). These covariates identified subjects at risk of failing to achieve a sorafenib Cmax ≥ 4.78 μg/mL with 95.0% specificity and 95.2% sensitivity. Concentration-guided sorafenib dosing with MIDS achieved a sorafenib Cmax within the range 4.78 to 5.78 μg/mL for 38 of 52 patients who failed to achieve a Cmax ≥ 4.78 μg/mL with standard dosing. In a simulation setting, concentration-guided dosing with MIDS was the quickest and most effective approach to achieve a sorafenib Cmax within a designated range.

Real-Life Experience of Sorafenib Maintenance after Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-ITD AML Reveals High Rates of Toxicity-Related Treatment Interruption

Introduction: FLT3-Internal Tandem Duplication (FLT3-ITD) mutations of the gene encoding the FLT3 tyrosine kinase receptor are found in 25-30% of AML patients and are associated with a poor prognosis despite intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). FLT3 tyrosine kinase inhibitors (TKI) significantly improve survival of FLT3 mutated AML patients in large prospective trials (Stone, NEJM 2017; doi:10.1056/NEJMoa1614359). Post-HSCT maintenance with sorafenib - a broad-spectrum TKI with strong activity against FLT3 - was recently shown to reduce the risk of relapse and improve survival in the phase II SORMAIN trial (Burchert, JCO 2020; doi: 10.1200/JCO.19.03345). Importantly, clinical trials reported a low rate of toxicities requiring sorafenib discontinuation (Chen et al, BBMT 2014;doi: 10.1016/j.bbmt.2014.09.007; Burchert et al., J Clin Oncol. 2020; doi: 10.1200/JCO.19.03345). The aim of our single-center retrospective analysis was to evaluate the real-life experience in patients treated with post-allogeneic HSCT sorafenib maintenance therapy for FLT3-ITD AML with a particular focus on tolerability and toxicity-related treatment interruption. Methods: We conducted a single-center retrospective study on 33 FLT3-ITD AML patients undergoing allogeneic HSCT in complete remission between 2013 and 2020, including 20 patients who received sorafenib maintenance and 13 who did not. Sorafenib was started at hematologic reconstitution at 200 mg BID and increased at 400 mg BID after a week in case of good tolerance. Treatment was maintained until two years if tolerated. In case of toxicity, the drug was either reduced to 200mg BID, or stopped. Among the 13 patients in the untreated group, 8 were transplanted between 2013 and 2016 before we started to routinely use sorafenib maintenance, and 5 were transplanted in 2017 or later. Among the latter, sorafenib maintenance was not started because of aGVHD and insufficient hematologic reconstitution (n=3), low-allelic ratio of FLT3-ITD (n=1), early death due to PTLD (n=1). Results: In the sorafenib group (n=20), median time from transplant to sorafenib initiation was 68 days (range 40-213). Overall, 16 patients (80%) experienced toxicities leading to dose reduction (4 patients, 20%) or interruption (12 patients, 60%). In these patients, the average time on sorafenib before toxicity occurrence was 98 days (range 1-717; only one patient stopped treatment after 1 day because of severe diarrhea). Most common toxicities were skin (7, grade II), gastrointestinal (5, grade II), and hematologic (5, grade II and III). One patient experienced uveitis (grade III) and pneumonia (grade IV), both resolutive after sorafenib interruption. One patient experienced severe hypertension (grade III), 1 had hepatitis (grade III) and another one a PRESS syndrome (grade IV) possibly related to sorafenib. Other causes of sorafenib interruption included FLT3-ITD negative relapse in 1 patient, relapse in 1 patient, and end of scheduled maintenance in 1 patient. Among the 12 patients who interrupted sorafenib because of toxicities, 3 patients were re-challenged with good tolerance in 2 cases, while 7 were switched to midostaurine. Among these 7, 4 completed the 2-y treatment and 3 interrupted midostaurine because of toxicities (2 patients experienced digestive intolerance and 1 pneumonia). The average time of sorafenib exposure in the whole sorafenib group was 180 days (range 1-765). Importantly, the analysis of patients' outcome confirmed the previously reported positive impact of sorafenib maintenance on overall survival (Figure 1B) despite high rates of treatment interruption. Conclusion: Our real-life analysis reveals higher rates of toxicity-related interruption of sorafenib maintenance after allogeneic HSCT than previously reported in clinical trials. Interestingly, our results confirm the favorable impact of sorafenib maintenance on clinical outcome despite a high rate of toxicity-related treatment interruption, suggesting the feasibility of switching to other maintenance approaches in case of low tolerability. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: sorafenib as a maintenance treatment to prevent FLT3-ITD mutated AML reccurence after allogeneic transplantation

Valproic acid overcomes sorafenib resistance by reducing the migration of Jagged2-mediated Notch1 signaling pathway in hepatocellular carcinoma cells.

Sorafenib resistance is a classic problem related to the treatment of advanced hepatocellular carcinoma (HCC). There is a recognized need to explore new drug resistance mechanisms and develop novel strategies to overcome the acquired resistance to sorafenib. Although one study has showed that the anti-epileptic drug valproic acid (VPA) could sensitize transforming growth factor-β (TGF-β)-induced sorafenib-resistant HCC cells, it is unclear whether VPA could reverse resistance to long-term clinical treatment with sorafenib. In this study, we successfully established sorafenib-resistant HCC cells by long-term sorafenib exposure. Compared with sensitive HCC cells, the proliferation, anti-apoptotic capability and migration of the sorafenib-resistant cells were enhanced. In addition, we found that VPA combined with sorafenib could overcome drug resistance by downregulating Jagged2-mediated Notch1 signaling pathway and epithelial-mesenchymal transition (EMT)-related proteins. Furthermore, the combination of VPA and sorafenib could obviously increase the sensitivity of drug-resistant cells in vitro and synergistically suppress tumor growth in vivo. These results provided a new insight that the use of VPA in combination with sorafenib was an effective method for clinically solving the problem of sorafenib resistance by modulating the Jagged2-mediated Notch1 signaling pathway and reversing the EMT phenotype.

Sorafenib exposure and its correlation with response and safety in advanced hepatocellular carcinoma: results from an observational retrospective study.

Severe adverse events frequently occur in patients treated with sorafenib, whereas some patients have suboptimal response to sorafenib. We aimed to evaluate the association of sorafenib-induced toxicities and clinical outcomes with the pharmacokinetics of sorafenib in patients with hepatocellular carcinoma (HCC). This was a retrospective, observational study in which 26 HCC patients who had been treated with sorafenib were enrolled between September 2010 and March 2015. The association between trough sorafenib concentration and occurrence of grade ≥ 3 toxicities was evaluated. In addition, we estimated the association of trough sorafenib concentration with overall survival (OS). The median sorafenib concentration was 2.91μg/mL (range 0.74-8.8μg/mL). Based on the receiver operating characteristic curve, the threshold value of the trough sorafenib concentration for predicting grade ≥ 3 toxicities and responder (complete response or partial response at best response, or stable disease for ≥ 3months) was 3.45μg/mL [area under the curve (AUC) 0.74, 95% confidence interval (CI) 0.54-0.93; p <0.05] and 1.40μg/mL (AUC 0.97, 95% CI 0.97-1.00; p <0.05), respectively. OS of patients with sorafenib 1.40-3.45µg/mL had a tendency to be longer than those of patients administered < 1.40μg/mL and ≥ 3.45μg/mL [median 17.8months (1.40-3.45μg/mL) vs. 5.3months (< 1.40μg/mL) and 9.5months (≥ 3.45μg/mL)]. From results of this study, we proposed that the target range of sorafenib may be a trough concentration of 1.40-3.45μg/mL in patients with HCC.

Hypoxia-Induced Aquaporin-3 Changes Hepatocellular Carcinoma Cell Sensitivity to Sorafenib by Activating the PI3K/Akt Signaling Pathway.

PurposeHypoxia-induced changes are primarily activated in patients with hepatocellular carcinoma (HCC) and long-term sorafenib exposure, thereby reducing the sensitivity to the drug. Aquaporin-3 (AQP3), a member of the aquaporin family, is a hypoxia-induced substance that affects the chemosensitivity of non-hepatocellular tumors. However, its expression and role in the sensitivity of hypoxic HCC cells to sorafenib-induced apoptosis remain unclear. The purpose of this study was to detect changes in AQP3 expression in hypoxic HCC cells and to determine whether these changes alter the sensitivity of these cells to sorafenib.Materials and MethodsHuh7 and HepG2 hypoxic cell models were established and AQP3 expression was detected using quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Furthermore, the role of AQP3 in cell sensitivity to sorafenib was evaluated via flow cytometry, Western blotting, and a CCK-8 assay.ResultsThe results of qPCR and Western blotting showed that AQP3 was overexpressed in the Huh7 and HepG2 hypoxic cell models. Furthermore, AQP3 protein levels were positively correlated with hypoxia-inducible factor-1α (HIF-1α) levels. Compared with cells transfected with lentivirus-GFP (Lv-GFP), hypoxic cells transfected with lentivirus-AQP3 (Lv-AQP3) were less sensitive to sorafenib-induced apoptosis. However, the sensitivity to the drug increased in cells transfected with lentivirus-AQP3RNAi (Lv-AQP3RNAi). Akt and Erk phosphorylation was enhanced in Lv-AQP3-transfected cells. Compared with UO126 (a Mek1/2 inhibitor), LY294002 (a PI3K inhibitor) attenuated the AQP3-induced insensitivity to sorafenib observed in hypoxic cells transfected with Lv-AQP3. Combined with LY294002-treated cells, hypoxic cells transfected with Lv-AQP3RNAi were more sensitive to sorafenib.ConclusionThe study results show that AQP3 is a potential therapeutic target for improving the sensitivity of hypoxic HCC cells to sorafenib.

Sorafenib administered using a high-dose, pulsatile regimen in patients with advanced solid malignancies: a phase I exposure escalation study

Background(Pre)clinical evidence is accumulating that intermittent exposure to increased doses of protein kinase inhibitors may improve their treatment benefit. In this phase I trial, the safety of high-dose, pulsatile sorafenib was studied.Patients and methodsHigh-dose sorafenib was administered once weekly in exposure escalation cohorts according to a 3 + 3 design. Drug monitoring was performed in weeks 1–3 and doses were adjusted to achieve a predefined target plasma area under the curve (AUC)(0–12 h). The effect of low gastric pH on improving sorafenib exposure was investigated by intake of the acidic beverage cola.ResultsSeventeen patients with advanced malignancies without standard treatment options were included. Once weekly, high-dose sorafenib exposure was escalated up to a target AUC(0–12 h) of 125–150 mg/L/h, achieving a twofold higher Cmax compared to standard continuous dosing. Dose-limiting toxicity was observed in three patients: grade 3 duodenal perforation (2800 mg sorafenib), grade 5 multiorgan failure (2800 mg sorafenib) and grade 5 biliary tract perforation (3600 mg sorafenib). The mean difference between observed and target AUC(0–12 h) was 45% (SD ± 56%) in week 1 using a fixed starting dose of sorafenib compared to 2% (SD ± 32%) in week 3 as a result of drug monitoring (P = 0.06). Dissolving sorafenib in cola, instead of water, did not improve sorafenib exposure. Clinical benefit with stable disease as the best response was observed in two patients.ConclusionTreatment with high-dose, once weekly sorafenib administration resulted in dose-limiting toxicity precluding dose escalation above the exposure cohort of 125–150 mg/L/h. Drug monitoring was a successful strategy to pursue a target exposure.

Cautious optimism-the current role of immunotherapy in gastrointestinal cancers.

Immunotherapy has been described as the "fourth pillar" of oncology treatment, in conjunction with surgery, chemotherapy, and radiotherapy. However, the role of immunotherapy in gastrointestinal tumours is still evolving. Data for checkpoint inhibition in esophagogastric, hepatocellular, colorectal, and anal squamous cell carcinomas are expanding. In phase iii trials in the second-line setting, PD-1 inhibitors have demonstrated positive results for the subset of esophageal cancers that are positive for PD-L1 at a combined positive score of 10 or more. Based on results of phase ii trials, PD-1 inhibitors were approved in North America for use in PD-L1-positive chemorefractory gastric cancers, in hepatocellular carcinoma after sorafenib exposure, and in treatment-refractory deficient mismatch repair (dmmr) or high microsatellite instability (msi-h) tumours, regardless of tissue site. Combination use of PD-1 and ctla-4 inhibitors has been approved by the U.S. Food and Drug Administration for chemorefractory dmmr or msi-h colorectal cancer. Responses to checkpoint inhibition are durable, particularly in the dmmr or msi-h colorectal cancer cohort. As trials of combination immunotherapy, immunotherapy in combination with other systemic therapies, and immunotherapy in combination with other treatment modalities move forward in multiple tumour sites, cautious optimism is called for. The treatment landscape is continually changing, and expanded indications are likely to be just around the corner.