Tyrosine Kinase Inhibitors Sorafenib Research Articles

Systemic Treatment of Recurrent Hepatocellular Carcinoma after Liver Transplantation: A Multicenter Trial.

The tyrosine kinase inhibitors (TKIs) sorafenib and lenvatinib represent the first-line systemic therapy of choice for patients with hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT). Under sorafenib and lenvatinib, HCC patients have shown increasingly improved overall survival in clinical studies over the years. In contrast, data on overall survival for patients with HCC recurrence after LT under TKIs are scarce and limited to small retrospective series. In this retrospective, multicenter study, we investigated the efficacy of TKI therapy and the influence of immunosuppression in patients with HCC recurrence after LT. Retrospective data were collected from four transplant centers from Germany and Austria. We included patients with HCC recurrence after LT between 2007 and 2020 who were treated with a TKI. In total, we analyzed data from 46 patients with HCC recurrence after LT. The most common underlying liver disease was hepatitis C, accounting for 52.2%. The median time to relapse was 11.8 months (range 0-117.7 months). The liver graft was affected in 21 patients (45.7%), and 36 patients (78.3%) had extrahepatic metastases at initial diagnosis of recurrence, with the lung being the most commonly affected (n = 25, 54.3%). Of the total, 54.3% (n = 25) of the patients were initially treated locally; 39 (85.8%) and 7 (15.2%) patients received sorafenib and lenvatinib, respectively, as first-line systemic therapy. Median overall survival of the whole cohort was 10.9 months (95% confidence interval (95% CI) 6.9-14.9 months) and median progression free survival was 5.7 months (95% CI 2.0-9.4 months) from treatment initiation. Since history of liver transplantation is considered a contraindication for immunotherapy, prognosis of patients with HCC recurrence after LT remains poor.

Anti-angiogenic and antioxidant effects of axitinib in human retinal endothelial cells: implications in diabetic retinopathy.

Diabetic retinopathy is a secondary microvascular complication of diabetes mellitus. This disease progresses from two stages, non-proliferative and proliferative diabetic retinopathy, the latter characterized by retinal abnormal angiogenesis. Pharmacological management of retinal angiogenesis employs expensive and invasive intravitreal injections of biologic drugs (anti-vascular endothelial growth factor agents). To search small molecules able to act as anti-angiogenic agents, we focused our study on axitinib, which is a tyrosine kinase inhibitor and represents the second line treatment for renal cell carcinoma. Axitinib is an inhibitor of vascular endothelial growth factor receptors, and among the others tyrosine kinase inhibitors (sunitinib and sorafenib) is the most selective towards vascular endothelial growth factor receptors 1 and 2. Besides the well-known anti-angiogenic and immune-modulatory functions, we hereby explored the polypharmacological profile of axitinib, through a bioinformatic/molecular modeling approach and in vitro models of diabetic retinopathy. We showed the anti-angiogenic activity of axitinib in two different in vitro models of diabetic retinopathy, by challenging retinal endothelial cells with high glucose concentration (fluctuating and non-fluctuating). We found that axitinib, along with inhibition of vascular endothelial growth factor receptors 1 (1.82 ± 0.10; 0.54 ± 0.13, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) and vascular endothelial growth factor receptors 2 (2.38 ± 0.21; 0.98 ± 0.20, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively), was able to significantly reduce (p < 0.05) the expression of Nrf2 (1.43 ± 0.04; 0.85 ± 0.01, protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in retinal endothelial cells exposed to high glucose, through predicted Keap1 interaction and activation of melanocortin receptor 1. Furthermore, axitinib treatment significantly (p < 0.05) decreased reactive oxygen species production (0.90 ± 0.10; 0.44 ± 0.06, fluorescence units in high glucose vs . axitinib 1 µM, respectively) and inhibited ERK pathway (1.64 ± 0.09; 0.73 ± 0.06, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in HRECs exposed to high glucose. The obtained results about the emerging polypharmacological profile support the hypothesis that axitinib could be a valid candidate to handle diabetic retinopathy, with ancillary mechanisms of action.

Selective therapeutic efficacy of tyrosine kinase inhibitor sorafenib on the restoration of methylglyoxal-induced peritoneal fibrosis

Peritoneal fibrosis, a common complication observed in long-term peritoneal dialysis patients, can gradually lead to ultrafiltration failure and the development of encapsulating peritoneal sclerosis. Although mechanisms of peritoneal fibrosis have been proposed, effective therapeutic options are unsatisfactory. Recently, several tyrosine kinase inhibitors have proven to be anti-fibrosis in rodent models. To assess the potential therapeutic effects of tyrosine kinase inhibitors on peritoneal fibrosis in the larger animal model, a novel porcine model of peritoneal fibrosis induced by 40 mM methylglyoxal in 2.5 % dialysate was established, and two different doses (20 mg/kg and 30 mg/kg) of sorafenib were given orally to evaluate their therapeutic efficacy in this study. Our results showed that sorafenib effectively reduced adhesions between peritoneal organs and significantly diminished the thickening of both the parietal and visceral peritoneum. Angiogenesis, vascular endothelial growth factor A production, myofibroblast infiltration, and decreased endothelial glycocalyx resulting from dialysate and methylglyoxal stimulations were also alleviated with sorafenib. However, therapeutic efficacy in ameliorating loss of mesothelial cells, restoring decreased ultrafiltration volume, and improving elevated small solutes transport rates was limited. In conclusion, this study demonstrated that sorafenib could potentially be used for peritoneal fibrosis treatment, but applying sorafenib alone might not be sufficient to fully rescue methylglyoxal-induced peritoneal defects.

Comprehensive analysis of differentially expressed miRNAs in hepatocellular carcinoma: Prognostic, predictive significance and pathway insights.

Robust prognostic and predictive factors for hepatocellular carcinoma, a leading cause of cancer-related deaths worldwide, have not yet been identified. Previous studies have identified potential HCC determinants such as genetic mutations, epigenetic alterations, and pathway dysregulation. However, the clinical significance of these molecular alterations remains elusive. MicroRNAs are major regulators of protein expression. MiRNA functions are frequently altered in cancer. In this study, we aimed to explore the prognostic value of differentially expressed miRNAs in HCC, to elucidate their associated pathways and their impact on treatment response. To this aim, bioinformatics techniques and clinical dataset analyses were employed to identify differentially expressed miRNAs in HCC compared to normal hepatic tissue. We validated known associations and identified a novel miRNA signature with potential prognostic significance. Our comprehensive analysis identified new miRNA-targeted pathways and showed that some of these protein coding genes predict HCC patients' response to the tyrosine kinase inhibitor sorafenib.

Induction of the Inflammasome Pathway by Tyrosine Kinase Inhibitors Provides an Actionable Therapeutic Target for Hepatocellular Carcinoma.

During the last decade, tyrosine kinase inhibitors (TKIs) sorafenib and regorafenib have been standard systemic treatments for advanced hepatocellular carcinoma (HCC). Previous data associated sorafenib with inflammasome activation. However, the role of the inflammasome in sorafenib and regorafenib signaling has not been described in liver cancer patients. For this purpose, we analyzed inflammasome-related transcriptomic changes in a murine HCC model. Our data confirmed inflammasome activation after both TKI treatments, sharing a similar pattern of increased gene expression. According to human database results, transcriptional increase of inflammasome genes is associated with poorer prognosis for male liver cancer patients, suggesting a sex-dependent role for inflammasome activation in HCC therapy. In biopsies of HCC and its surrounding tissue, we detected durable increases in the inflammasome activation pattern after sorafenib or regorafenib treatment in male patients. Further supporting its involvement in sorafenib action, inflammasome inhibition (MCC950) enhanced sorafenib anticancer activity in experimental HCC models, while no direct in vitro effect was observed in HCC cell lines. Moreover, activated human THP-1 macrophages released IL-1β after sorafenib administration, while 3D Hep3B spheres displayed increased tumor growth after IL-1β addition, pointing to the liver microenvironment as a key player in inflammasome action. In summary, our results unveil the inflammasome pathway as an actionable target in sorafenib or regorafenib therapy and associate an inflammasome signature in HCC and surrounding tissue with TKI administration. Therefore, targeting inflammasome activation, principally in male patients, could help to overcome sorafenib or regorafenib resistance and enhance the efficacy of TKI treatments in HCC.

Standard therapy or additionally radioactive iodine (131I) therapy; which will stop the recurrence of glioblastoma multiforme (GBM)?

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour. The average survival time for a patient diagnosed with GBM, using standard treatment methods, is several months. Authors of the article pose a direct question: Is it possible to treat GBM solely with radioactive iodine (¹³¹I) therapy without employing the sodium iodide symporter (NIS) gene? After all, NIS has been detected not only in the thyroid but also in various tumours. The main author of this article (A.C.), with the assistance of her colleagues (physicians and pharmacologists), underwent ¹³¹I therapy after prior iodine inhibition, resulting in approximately 30% reduction in tumour size as revealed by magnetic resonance imaging (MRI). Classical therapy for GBM encompasses neurosurgery, conventional radiotherapy, and chemotherapy (e.g. temozolomide). Currently, tyrosine kinase inhibitors (imatinib, sunitinib, and sorafenib) are being used. Additionally, novel drugs such as crizotinib, entrectinib, or larotrectinib are being applied. Recently, personalised multimodal immunotherapy (IMI) based on anti-tumour vaccines derived from oncolytic viruses has been developed, concomitant with the advancement of cellular and molecular immunology. Thus, ¹³¹I therapy has been successfully employed for the first time in the case of GBM recurrence.

Low-Dose Sorafenib Promotes Cancer Stem Cell Expansion and Accelerated Tumor Progression in Soft Tissue Sarcomas.

The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients.

Selective expansion of renal cancer stem cells using microfluidic single-cell culture arrays for anticancer drug testing.

The suboptimal prognosis associated with drug therapy for renal cancer can be attributed to the presence of stem-cell-like renal cancer cells. However, the limited number of these cells prevents conventional drug screening assays from effectively assessing the response of renal cancer stem cells to anti-cancer agents. To address this issue, the present study employed microfluidic single-cell culture arrays to expand renal cancer stem cells by exploiting the anti-apoptosis and self-renewal properties of tumor stem cells. A microfluidic chip with 18 000 hydrophilic microwells was designed and fabricated to establish the single-cell culture array. Over a 7 day culture, the large-scale single-cell culture yielded a limited quantity of single-cell-derived tumorspheres. The sphere formation rates for Caki-1, 786-O, and ACHN cells were determined to be 8.74 ± 0.53%, 12.02 ± 1.43%, and 4.98 ± 1.68%, respectively. The expanded cells exhibited stemness characteristics, as indicated by immunofluorescence, flow cytometry, serial passaging, and in vitro differentiation assays. Additionally, the comparative transcriptomic analysis showed significant differences in the gene expression patterns of the expanded cells compared to the differentiated renal cancer cells. The drug testing indicated that renal cancer stem cells exhibited reduced sensitivity towards the tyrosine kinase inhibitors sorafenib and sunitinib, compared to differentiated renal cancer cells. This reduced sensitivity can be attributed to the elevated expression levels of tyrosine kinase in renal cancer stem cells. This present study provides evidence that the utilization of microfluidic single-cell culture arrays for selective cell expansion can facilitate drug testing of renal cancer stem cells.

SHP2 Inhibition Overcomes Adaptive and Acquired Resistance to FLT3 TKI to Improve Efficacy Against FLT3/ITD AML

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase (RTK) that is frequently mutated in acute myeloid leukemia (AML), with the internal tandem mutation (FLT3/ITD) conferring a poor prognosis to AML patients. This makes FLT3/ITD AML a validated target for treatment with tyrosine kinase inhibitors (TKI), but their success has been hindered by mechanisms of resistance including rapid signal reprogramming known as adaptive resistance and acquired resistance gained through long-term treatment. Past work from our lab has shown that FLT3/ITD cells undergo adaptive resistance, through the rapid reactivation of ERK signaling within 24 hours of sustained FLT3 inhibition. To improve therapy of FLT3/ITD AML it is important to identify therapeutic targets that overcome this ERK reactivation. Since adaptive ERK reactivation is often due to the relief of negative feedback loops that result in the activation of other RTK, we performed an RTK phospho-array on the FLT3/ITD cell line MV4;11. 48-hour treatment with the FLT3 TKI sorafenib resulted in increased activation of IGF-R, HER3 and AXL. While recent work from our lab found that combining AXL and FLT3 inhibition abrogated the ERK reactivation and exerted greater antileukemic effects, the activation of multiple RTKs suggests that targeting a signaling hub that inhibits multiple RTKs at once will likely prove more efficacious in overcoming this adaptive response. Recent literature has implicated SH2-containing protein tyrosine phosphatase 2 (SHP2) as a therapeutic target in adaptive ERK reactivation in other cancers. SHP2 acts directly downstream of RTKs and is involved in the activation of the ERK pathway. Therefore, we explored whether the addition of an SHP2 inhibitorto FLT3 TKI can overcome the adaptive response and increase treatment efficacy. To explore this possibility, we first treated the FLT3/ITD cell lines Molm14 and MV4;11 as well as FLT3/ITD patient samples with FLT3 TKI alone or in combination with an SHP2 inhibitor for 24 hours. The addition of SHP099 to sorafenib decreased the expected ERK reactivation in a dose-dependent manner. Furthermore, combination treatment with synergized to decrease proliferation and increase apoptosis in the FLT3/ITD cell lines. Impressively, in a long-term assay, treatment with the combination for just 72 hours greatly reduced leukemic cell recovery. To determine whether this combination would improve efficacy in vivo, we engrafted NSG mice with Molm14 cells and treated them for 4 weeks via oral gavage. The combination of SHP099 with gilteritinib group resulted in a statistically significantly lower leukemia burden compared to either treatment alone and the vehicle control. The combination group also had prolonged survival compared to the other groups. In addition to rapid adaptive resistance, there are mechanisms of acquired resistance that render traditional FLT3 TKI treatments ineffective against FLT3/ITD AML cells. We next examined the effects of SHP2 inhibition on FLT3-TKI resistant cells models that were generated by growing and maintaining Molm14 and MV4;11 cells in the presence of FLT3 TKI. The combination of FLT3 TKI and SHP099 resensitized the cells to FLT3 TKI, resulting in decreased ERK phosphorylation, decreased proliferation and increased apoptosis of these FLT3 TKI resistant cells. We also explored the effects of treating the FLT3/ITD mutant cell lines with the combination of SHP099 and several of the cytotoxic chemotherapy agents typically used in the treatment of AML including cytarabine and daunorubicin. While daunorubicin or cytarabine alone had little effect on the level of phosphorylated ERK, the addition of SHP099 decreased ERK activation and the combination synergized to exert greater to decrease proliferation and increase apoptosis. These studies reveal that combining FLT3 TKI treatment with an allosteric SHP2 inhibitor decreases the adaptive ERK reactivation observed in FLT3/ITD cells. The finding that SHP099 synergized with both FLT3 TKI and chemotherapy agents in different FLT3-mutated AML models speaks to the versatile efficacy of SHP2 inhibition in multiple AML models. Taken together, the data suggests that SHP2 inhibition can help overcome both adaptive and acquired resistance in FLT3/ITD AML and is a candidate to try to improve patient outcomes.

Combination therapy of tyrosine kinase inhibitor sorafenib with the HSP90 inhibitor onalespib as a novel treatment regimen for thyroid cancer

Thyroid cancer is the most common endocrine malignancy, affecting nearly 600,000 new patients worldwide. Treatment with the BRAF inhibitor sorafenib partially prolongs progression-free survival in thyroid cancer patients, but fails to improve overall survival. This study examines enhancing sorafenib efficacy by combination therapy with the novel HSP90 inhibitor onalespib. In vitro efficacy of sorafenib and onalespib monotherapy as well as in combination was assessed in papillary (PTC) and anaplastic (ATC) thyroid cancer cells using cell viability and colony formation assays. Migration potential was studied in wound healing assays. The in vivo efficacy of sorafenib and onalespib therapy was evaluated in mice bearing BHT-101 xenografts. Sorafenib in combination with onalespib significantly inhibited PTC and ATC cell proliferation, decreased metabolic activity and cancer cell migration. In addition, the drug combination approach significantly inhibited tumor growth in the xenograft model and prolonged the median survival. Our results suggest that combination therapy with sorafenib and onalespib could be used as a new therapeutic approach in the treatment of thyroid cancer, significantly improving the results obtained with sorafenib as monotherapy. This approach has the potential to reduce treatment adaptation while at the same time providing therapeutic anti-cancer benefits such as reducing tumor growth and metastatic potential.

Potential Predictive Biomarkers of Systemic Drug Therapy for Hepatocellular Carcinoma: Anticipated Usefulness in Clinical Practice.

In the systemic drug treatment of hepatocellular carcinoma, only the tyrosine kinase inhibitor (TKI) sorafenib was available for a period. This was followed by the development of regorafenib as a second-line treatment after sorafenib, and then lenvatinib, a new TKI, proved non-inferiority to sorafenib and became available as a first-line treatment. Subsequently, cabozantinib, another TKI, was introduced as a second-line treatment, along with ramucirumab, the only drug proven to be predictive of therapeutic efficacy when AFP levels are >400 ng/mL. It is an anti-VEGF receptor antibody. More recently, immune checkpoint inhibitors have become the mainstay of systemic therapy and can now be used as a first-line standard treatment for HCC. However, the objective response rate for these drugs is currently only 30% to 40%, and there is a high incidence of side effects. Additionally, there are no practical biomarkers to predict their therapeutic effects. Therefore, this review provides an overview of extensive research conducted on potential HCC biomarkers from blood, tissue, or imaging information that can be used in practice to predict the therapeutic efficacy of systemic therapy before its initiation.

Effects of Lenvatinib on Skeletal Muscle Volume and Cardiac Function in Patients with Hepatocellular Carcinoma

Introduction: Previously, we reported that the tyrosine kinase inhibitor (TKI) sorafenib decreases serum levels of carnitine and reduces skeletal muscle volume. Moreover, others reported that TKIs might lead to cardiomyopathy or heart failure. Therefore, this study aimed to evaluate the effects of lenvatinib (LEN) on skeletal muscle volume and cardiac function in patients with hepatocellular carcinoma (HCC). Methods: This retrospective study included 58 adult Japanese patients with chronic liver diseases and HCC treated with LEN. Blood samples were collected before and after 4 weeks of treatment, and serum carnitine fraction and myostatin levels were measured. Before and after 4–6 weeks of treatment, the skeletal muscle index (SMI) was evaluated from computed tomography images and cardiac function was assessed by ultrasound cardiography. Results: After treatment, SMI, serum levels of total carnitine, and global longitudinal strain were significantly lower, but serum levels of myostatin were significantly higher. Left ventricular ejection fraction showed no significant change. Conclusion: In patients with HCC, LEN decreases serum levels of carnitine, skeletal muscle volume, and worsens cardiac function.

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined. MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib. ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability. ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.

Angiogenesis and Hepatocellular Carcinoma: From Molecular Mechanisms to Systemic Therapies.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The hypervascular nature of the majority of HCCs and the peculiar vascular derangement occurring during liver carcinogenesis underscore the importance of angiogenesis in the development and progression of these tumors. Indeed, several angiogenic molecular pathways have been identified as deregulated in HCC. The hypervascular nature and the peculiar vascularization of HCC, as well as deregulated angiogenic pathways, represent major therapeutic targets. To a large extent, intra-arterial locoregional treatments (transarterial-(chemo)embolization) rely on tumor ischemia caused by embolization of tumor feeding arteries, even though this may represent the "primum movens" of tumor recurrence through the activation of neoangiogenesis. Considering systemic therapies, the currently available tyrosine kinase inhibitors (sorafenib, regorafenib, cabozantinib and lenvatinib) and monoclonal antibodies (ramucirumab and bevacizumab, in combination with the anti-PD-L1, atezolizumab) primarily target, among others, angiogenic pathways. Considering the importance of angiogenesis in the pathogenesis and treatment of liver cancer, in this paper, we aim to review the role of angiogenesis in HCC, addressing the molecular mechanisms, available antiangiogenic therapies and prognostic biomarkers in patients receiving these treatments.

The Molecular Pharmacology and Therapeutics of Angiogenesis

Angiogenesis is the formation of new vasculature on top of the pre-existing ones, which is critical during the species’ lifespan. It is responsible for delivering oxygen and nutrients to metabolically active tissues, therefore playing an essentialrole in physiological conditions, such as wound healing and pathological circumstances, like malignancies. Althoughsharing similar characteristics, the blood vessels in pathological angiogenesis develop irregularly and disorganizedly.Therefore, the leakage of materials from blood vessels is familiar in abnormal angiogenesis, which may result inmuch higher interstitial pressure than internal pressure. Whether the angiogenesis is physiological or pathological,the proliferation of normal dormant endothelial cells is an essential requirement. Additionally, angiogenesis is alsoinitiated by vascular endothelial growth factor A (VEGF-A) interacting with the vascular endothelial growth factorreceptor 2 (VEGFR2) or the co-receptor neuropilin-1 (NRP-1). The Vegfa gene can be spliced in many ways, creatingnumerous VEGF-A isoforms, each with unique interactions with VEGFR2. Several drugs have been approved as thefirst- or second-line of angiogenesis, thereby preventing cancer progression. The widely used is monoclonal antibodies(bevacizumab) and receptor tyrosine kinase inhibitor (sorafenib and axitinib). However, they lack selectivity and, asa result, are less effective and have considerable adverse effects. As a natural drug, Paclitaxel has an anti-angiogeniceffect in addition to its chemotherapeutic effect. However, different doses of paclitaxel have different anti-angiogenicmechanisms and different effects on different species.

The Molecular Pharmacology and Therapeutics of Angiogenesis

Angiogenesis is the formation of new vasculature on top of the pre-existing ones, which is critical during the species’ lifespan. It is responsible for delivering oxygen and nutrients to metabolically active tissues, therefore playing an essentialrole in physiological conditions, such as wound healing and pathological circumstances, like malignancies. Althoughsharing similar characteristics, the blood vessels in pathological angiogenesis develop irregularly and disorganizedly.Therefore, the leakage of materials from blood vessels is familiar in abnormal angiogenesis, which may result inmuch higher interstitial pressure than internal pressure. Whether the angiogenesis is physiological or pathological,the proliferation of normal dormant endothelial cells is an essential requirement. Additionally, angiogenesis is alsoinitiated by vascular endothelial growth factor A (VEGF-A) interacting with the vascular endothelial growth factorreceptor 2 (VEGFR2) or the co-receptor neuropilin-1 (NRP-1). The Vegfa gene can be spliced in many ways, creatingnumerous VEGF-A isoforms, each with unique interactions with VEGFR2. Several drugs have been approved as thefirst- or second-line of angiogenesis, thereby preventing cancer progression. The widely used is monoclonal antibodies(bevacizumab) and receptor tyrosine kinase inhibitor (sorafenib and axitinib). However, they lack selectivity and, asa result, are less effective and have considerable adverse effects. As a natural drug, Paclitaxel has an anti-angiogeniceffect in addition to its chemotherapeutic effect. However, different doses of paclitaxel have different anti-angiogenicmechanisms and different effects on different species.

The Molecular Pharmacology and Therapeutics of Angiogenesis

Angiogenesis is the formation of new vasculature on top of the pre-existing ones, which is critical during the species’ lifespan. It is responsible for delivering oxygen and nutrients to metabolically active tissues, therefore playing an essentialrole in physiological conditions, such as wound healing and pathological circumstances, like malignancies. Althoughsharing similar characteristics, the blood vessels in pathological angiogenesis develop irregularly and disorganizedly.Therefore, the leakage of materials from blood vessels is familiar in abnormal angiogenesis, which may result inmuch higher interstitial pressure than internal pressure. Whether the angiogenesis is physiological or pathological,the proliferation of normal dormant endothelial cells is an essential requirement. Additionally, angiogenesis is alsoinitiated by vascular endothelial growth factor A (VEGF-A) interacting with the vascular endothelial growth factorreceptor 2 (VEGFR2) or the co-receptor neuropilin-1 (NRP-1). The Vegfa gene can be spliced in many ways, creatingnumerous VEGF-A isoforms, each with unique interactions with VEGFR2. Several drugs have been approved as thefirst- or second-line of angiogenesis, thereby preventing cancer progression. The widely used is monoclonal antibodies(bevacizumab) and receptor tyrosine kinase inhibitor (sorafenib and axitinib). However, they lack selectivity and, asa result, are less effective and have considerable adverse effects. As a natural drug, Paclitaxel has an anti-angiogeniceffect in addition to its chemotherapeutic effect. However, different doses of paclitaxel have different anti-angiogenicmechanisms and different effects on different species.

Comprehensive CRISPR-enabled functional genomics profiling to identify targetable cancer genes in primary acute myeloid leukemia (AML).

3088 Background: Acute myeloid leukemia (AML) is a life-threatening hematopoietic malignancy affecting both older and younger patient populations. Despite the approval of multiple new therapeutics during the past decade, including new targeted agents, treatment outcomes for AML remain poor, with &lt; 10% 5-year survival in patients over 60 years of age, and &lt; 50% 5-year survival in younger patients. Also, comprehensive genomic profiling (CGP) for common molecular lesions (e.g., FLT3 mutation) incompletely stratifies the patient population, indicating that a more comprehensive approach to personalized medicine could be of significant clinical benefit. To this end we developed Fx Heme, an in vitro functional genomics platform using CRISPR/Cas9 technology in primary cancer patient samples. Previously, we have shown it to stratify clinical response in AML patients treated with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g., GCLAM). Here we profile an expanded cohort to systematically characterize the AML functional genomics landscape and identify potential new targets for AML drug development. Methods: Unsorted primary tumor cells from patient samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library which was designed to inhibit expression of genes encoding the targets of FDA-approved targeted oncology drugs, as well as internal negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated for each gene to generate gene-level statistics, enabling identification of gene dependencies. Results: Comprehensive CRISPR/Cas9 functional genomics profiling was carried out retrospectively on pre-therapy samples from 59 AML patients. There were no false positives identified among negative controls. Functional dependencies on multiple genes were observed, with approximately half of patients (n=31; 52.5%) showing at least one gene dependency. We observed on average 6.7 dependencies per positive sample, with 5 of them revealing more than 10 dependencies. In addition to the targets of drugs approved to treat AML (FLT3, 10.2%; IDH1/2, 3.4%; SMO, 3.4%), a diversity of targetable genes for new therapeutic approaches to AML were identified across multiple known oncogenic signaling pathways. The prevalence of these recurrent gene dependencies ranged from 3.3% to 23.7% in our AML cohort. Conclusions: In summary, comprehensive functional genomic profiling using Fx Heme provides an effective platform to identify therapeutic opportunities in AML based on dependencies identified for targets of drugs not currently used to treat AML. These insights are now being utilized to develop additional treatment options and more effective personalization of treatment for AML.

Abstract LB192: MTL-CEBPA in combination with pembrolizumab converts an immune desert to an inflamed TME in solid tumors resistant to checkpoint blockade

Abstract Despite significant advances in outcomes with immunotherapy, most cancer patients do not benefit from currently approved immune checkpoint inhibitors (ICI). The reasons for ICI resistance are multi-faceted and suggest that additional immunomodulation is required to improve outcomes. MTL-CEBPA is a novel immunotherapy based on RNA activation that upregulates expression of a master myeloid transcription factor, CEBPA. The small activating RNA for CEBPA is encapsulated within a NOV340 liposome that targets the myeloid cell lineage. MTL-CEBPA has shown favorable safety and promising clinical activity in combination with tyrosine kinase inhibitors (Sorafenib) in hepatocellular carcinoma (NCT-02716012) [Hashimoto et al, CCR 2021; Sarker et al, CCR 2020]. We recently reported preliminary clinical data from the ongoing multi-center phase 1 TIMEPOINT study (NCT-04105335) evaluating the safety, pharmacokinetics, immunomodulation, and clinical activity of MTL-CEBPA in combination with pembrolizumab in patients with solid tumors who have exhausted standard therapy. This demonstrated a favorable safety profile and initial clinical activity [Plummer et al, JITC 2021]. Here we report the findings from a biomarker pharmacodynamic analysis of paired baseline and cycle 2 tumor sample biopsies in 23 patients from the TIMEPOINT trial. Brightplex® IHC and digital pathology analyses of the samples for myeloid and T cell panels were undertaken, alongside gene expression (Nanostring I/O 360). Prior to study treatment, nine patients out of 23 had an immune cold tumor microenvironment (TME) at baseline as measured by the Immunosign®21 score. Following the combination of MTL-CEBPA with pembrolizumab, seven of these patients converted to an inflamed TME by Immunosign®21 (P=0.008). This change in the TME was associated with infiltration of CD8 and cytotoxic T cells (CD8+, GrzB+, Ki-67+) (P=0.1). GSEA analysis indicated that a Tstem-like signature was enriched post-treatment. A Brightplex® IHC analysis of myeloid cells in these patients indicated that, post treatment, there was a significant influx of HLA-DR+ myeloid cells into the TME (P=0.04). We also observed a significant increase in the expression of CXCL9, 10, and 11. The remaining 14 patients had an inflamed TME at baseline. Here, we also observed an increase in HLA-DR+ cells, T cells, and chemokines, though to a lesser extent. Further, however, in these inflamed tumors—which have significantly greater infiltration of myeloid-derived suppressor cells (MDSCs) than desert tumors—we observed a reduction in 8/10 patients with detectable PMN-MDSCs (P=0.1) post treatment, consistent with the mechanism of action of CEBPA. An expression signature based on 18 genes significantly enriched for clinical response across all patients. Collectively, these data suggest a positive immunomodulatory TME effect of the combination of MTL-CEBPA with pembrolizumab. In both hot and cold TME tumors, the combination drives directed differentiation of progenitor monocytes into HLA-DR+ myeloid cells secreting chemokines that stimulate the ingress of T cells into the TME. We observe a significant positive correlation between the change in cytotoxic T cells and HLA-DR+ myeloid cells post treatment (P=0.004). These effects are most pronounced in cold tumors. Citation Format: Ruth Plummer, Mikael Sodergren, Brid Ryan, Ilian Tchakov, Nina Raulf, Rose Hodgson, CP Tan, Joanna P. Nicholls, Alison Adderkin, N Vasileiadou, Vikash Reebye, Tim Meyer, David J. Pinato, Debashis Sarker, Bristi Basu, Sarah Blagden, Natalie Cook, Jeff Evans, Jeffrey Yachnin, Cheng Ean Chee, Dan Li, Anthony El-Khoueiry, Maria Diab, Kai-Wen Huang, Marcus S. Noel, Bridget Keenan, Devalingam Mahalingam, Melanie Grosso, Denis Arnaud, Aurelie Auguste, Jan Storkholm, Iain McNeish, Robert Habib, John J. Rossi, Nagy Habib. MTL-CEBPA in combination with pembrolizumab converts an immune desert to an inflamed TME in solid tumors resistant to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB192.

Abstract 1062: A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme

Abstract AML is the most common acute leukemia in adults over 50 years of age. Current therapeutic approaches are focused on high or low dose chemotherapy in the front line setting, with use of molecularly targeted agents in unfit patients or the relapsed/refractory setting. However, routine genomic profiling for common molecular lesions (e.g. FLT3 mutation) incompletely stratifies the target patient population, and more personalized and comprehensive approaches could be of significant clinical benefit. To this end, we have developed an in vitro functional genomics platform for the systematic characterization of gene dependency on drug targets using CRISPR/Cas9 technology. Methods: Retrospective pre-therapy samples were obtained for a cohort of AML patients who subsequently went onto therapy with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g. GCLAM). Unsorted primary tumor cells from these samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library. This customized sgRNA library (Fx Heme) was designed to inhibit expression of genes encoding the targets of all FDA-approved oncology drugs, as well as internal positive and negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Dependency on targets of sorafenib was compared to clinical outcome (complete response (CR) without minimal residual disease (MRD), incomplete response, CR with MRD, or refractory) following treatment with sorafenib plus chemotherapy. Results: Among the 22 AML patients analyzed, 14 reached CR and 8 were classified as incomplete responders (refractory or MRD). Gene dependency on sorafenib targets including FLT3, KIT, PDGFR, and all RAF isoforms was frequently observed (12/22 cases). Among cases indicating any sorafenib target gene dependency, 5/12 (42%) demonstrated dependence on 2 or more sorafenib target genes. Overall, Fx Heme achieved 78.6% sensitivity, 87.5% specificity, and 91.7% PPV for prediction of observed clinical outcome. In contrast, stratification based on FLT3 mutation (internal tandem duplication or tyrosine kinase domain mutation) demonstrated 64.3% sensitivity, 50% specificity, 69.2% PPV. In summary, our results establish feasibility of CRISPR-based Fx Heme comprehensive functional genomic profiling for AML precision medicine, and validate this approach in a retrospective study cohort. Functional genomics may contribute to more effective personalization of AML treatment by uncovering drug target dependencies not readily identified by conventional genomic profiling, in an unbiased, comprehensive and patient-specific fashion. Citation Format: Stephane Corbel, Karl Hodel, Kathleen Tran, Maci Meyers, Jing Tong, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath. A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1062.