Targeted Kinase Inhibitors Research Articles

Abstract 3265: Secretome core fucosylation drives clonal drug resistance expansion and hypoxic control of metastasis

Abstract Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global secretome and PON1-specific de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. In a spontaneous lung metastasis mouse model, global secretome and PON1-specific core fucosylation functionally drive hypoxia and hypoxic selection of drug-resistant clones. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance and metastasis. Citation Format: Mark Borris D. Aldonza, Keun Hong Son, Je-Yoel Cho. Secretome core fucosylation drives clonal drug resistance expansion and hypoxic control of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3265.

Nintedanib downregulates the profibrotic M2 phenotype in cultured monocyte-derived macrophages obtained from systemic sclerosis patients affected by interstitial lung disease

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.MethodsFourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients’ informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFβ1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.ResultsCultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).ConclusionsIn cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFβ1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib invitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both invitro and invivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

Cabozantinib plus pembrolizumab as first-line therapy for cisplatin-ineligible advanced urothelial carcinoma (PemCab).

539 Background: Pembrolizumab (pem) was initially approved in the first line setting for cisplatin-ineligible metastatic urothelial carcinoma (mUC) patients (pts) with high PD-L1 expression and platinum ineligible pts regardless of PD-L1 status; this later changed to only platinum ineligible pts. There is a high disease progression (PD) rate with pem monotherapy. Cabozantinib (cabo) is a multiple receptor tyrosine kinase inhibitor targeting MET and VEGFR2 approved in multiple other malignancies as monotherapy or in combination with PD-1 inhibition. We hypothesized that combination of pem and cabo therapy will be safe and efficacious in patients with treatment naïve mUC. Methods: This is a phase II, open label, multi-center, single arm trial evaluating the tolerability and activity of pem 200 mg every 3 weeks and cabo 40 mg daily as first-line treatment for patients with mUC. Key inclusion criteria were histologically proven locally advanced or mUC, ECOG-PS 0-2, cisplatin-ineligible (including patient refusal of cisplatin), treatment naïve, and no prior PD-1/L1 therapy. The primary endpoint was objective response rate (ORR). Key secondary endpoints included progression-free survival (PFS) and overall survival (OS). There was a lead-in safety cohort of 6 pts. We hypothesized that combination therapy would improve ORR to &gt; 32%. With 35 evaluable subjects, the lower bound of the 95% confidence interval would extend no more than 26% from the observed proportion. With ≥17 objective responses, the confidence interval excludes 32%. Results: Between December 26, 2018, and April 19, 2023, 36 pts were enrolled of which 35 were evaluable for response with the median follow up of 14.4 months (95% CI 12.2 – 16.2). The median age was 72.5y [range, 47-82]; ECOG PS 0-1 in 89% pts. Responses were observed in 15 pts (ORR= 42.8%) including complete responses in 5 pts (14.3%). Stable disease was seen in 10 pts (28.6%). The disease control rate was 68.5% (25/35). Median PFS was 7.7 months (95% CI 4.2 – 11.2). Fifty-two percent of pts developed any grade treatment-related adverse events (TRAE) attributable to either cabozantinib or pembrolizumab. The most common grade 1/2 TRAE were diarrhea, anorexia, dysgeusia, weight loss, and nausea. Forty-four percent pts developed grade 3/4 TRAEs most common being hypertension, hypophosphatemia, alanine transaminase elevation, diarrhea, and fatigue. Conclusions: This novel phase II trial of pem + cabo demonstrated a manageable toxicity profile and promising efficacy as first-line therapy in mUC including patient’s ineligible for cisplatin. Further investigation with a focus on predictive biomarkers is ongoing. Clinical trial information: NCT03534804 .

Preliminary results of surufatinib (Suru) plus standard chemotherapy (ChT) as second line (2L) treatment of advanced gastric cancer (aGC): A single-arm, phase 2 clinical trial.

353 Background: Limited options are available for aGC patients who have failed first line (1L) systemic treatment, and single-agent ChT with paclitaxel (Pac) or irinotecan (Iri) remains the mainstay. Recent studies have suggested improved efficacy with the combination of ChT and antiangiogenic agents in the 2L setting. Suru is a tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1 and CSF-1R. This trial is aimed to evaluate the efficacy and safety of Suru plus Pac or Iri as 2L treatment for aGC. Methods: This single-arm, single center, phase 2 trial was designed to enroll up to 35 histologically confirmed HER2-negative aGC patients with evaluable disease who failed 1L ChT with or without immunotherapy (IO). Eligible patients were administered 21-day cycles of Suru (250mg, po, qd) plus Pac (150mg/m2, iv, d1) or Iri (125mg/m2, iv, d1, d8) per investigator choice for 6 cycles, followed by Suru (250mg, po, qd) maintenance if no disease progression (PD), until intolerable toxicity, PD, or death. The primary endpoint was ORR (per RECIST v1.1), and secondary endpoints included DCR, PFS, OS, and safety. Results: As of Sep 11, 2023, 15 patients were enrolled, and 6 patients remained on treatment. The median follow-up time was 5.77 (95% CI: 3.37~10.13) months. Among all 15 patients, the median age was 66 (range: 33-76) years, 14 (93.3%) were male, and 14 (93.3%) had an ECOG PS of 1. Ten (66.7%) patients had received prior anti-PD-1 antibodies, and all 15 patients received Pac as the standard ChT. All 15 patients were evaluable for tumor response, with 5 PR, 9 SD, and 1 PD as their best overall response (BOR). The ORR and DCR were 33.3% and 93.3%, respectively. The median PFS was 5.23 months, while the OS data was immature yet. The ORR, DCR and PFS seemed comparable between patients with and without prior IO. In terms of safety, the combined treatment was generally well tolerated, treatment-related Grade 3/4 adverse events included neutropenia (6, 40%), leukopenia (1, 6.7%), lymphopenia (1, 6.7%), and proteinuria (1, 6.7%). There were no treatment-related serious adverse events or treatment-related deaths. Conclusions: These results suggested promising anti-tumor activity and a manageable safety profile of Suru plus standard ChT in 2L treatment of aGC. The trial is still ongoing, and more data will be disclosed in the future. Clinical trial information: ChiCTR2200063336 . [Table: see text]

A phase II study to evaluate the safety and efficacy of anlotinib combined with penpulimab for advanced refractory biliary tract cancer.

TPS582 Background: Although with modest efficacy, mFOLFOX is recommended as standard second-line chemotherapy for advanced biliary tract cancer (BTC). Several clinical trials are exploring the combination treatment of antiangiogenic drugs and immune checkpoint inhibitors. Anlotinib is an oral multi-targeted tyrosine kinase inhibitor targeting VEGFR1/2/3, FGFR1-4 and PDGFRα/β, which effectively blocks tumor neovascularization and growth. Previous phase I/II clinical trials suggested that anlotinib combined with PD-(L)1 inhibitors as second-line therapy was well tolerated and showed clinical anti-tumor activity in advanced biliary tract cancer (NCT03825705, NCT03996408, ChiCTR1900022003, ChiCTR2000037847). Penpulimab is a novel humanized anti-PD-1 IgG1 antibody with complete removal of Fc receptor mediated effect, and featuring slow antigen binding off-rate and high receptor occupancy. In this study, we explore anlotinib plus penpulimab as a chemo-free combination for second-line and above therapy. Methods: This is a prospective, single-arm, phase II study. BTC patients (pts) failed after the first-line treatment, aged 18-75 and an ECOG PS of 0-1 were recruited.Eligible pts received anlotinib (12mg, po, d1-14, q3w) and penpulimab (200mg, iv, d1, q3w) until disease progression, unacceptable toxicity or up to 2 years. The primary endpoint was Objective Response Rate (ORR). Secondary endpoints included Overall survival (OS), Progression-Free Survival (PFS), Disease Control Rate (DCR) and safety. Based on a two-sided test for one proportion with 5.0% type I error, 80% power to detect an improvement in ORR from 5% to 22%, there will be 27 pts enrolled considering 20% of pts dropping out. Clinical trial information: ChiCTR2300069126 .

Surufatinib plus sintilimab in patients with advanced gastric/gastroesophageal junction adenocarcinoma (GC/GEJC): A single-arm, open-label, single-center phase II trial.

358 Background: Anti-PD-1 antibodies plus chemotherapy have become the standard first-line treatment for advanced GC/GEJC with CPS score ≥ 5. However, GC/GEJC still has dismal survival. Preclinical studies have demonstrated that surufatinib (a novel small-molecule kinase inhibitor targeting VEGFR1-3, FGFR and CSF-1R) combined with anti-PD-1 antibodies have synergistic antitumor effects by modulating tumor immune microenvironment. Therefore, a phase II trial to evaluate surufatinib plus sintilimab in patients (pts) with advanced GC/GEJC was conducted. Here we report the results of part A of this trial. Methods: This single arm, open-label, single center phase II trial consisted of two parts: part A (second-line therapy) and part B (first-line therapy). In part A, pts aged 18-75 years who were HER2-negative, failed first-line standard therapy, and immune checkpoint inhibitor therapy naive were enrolled. Part A included safety run-in and dose expansion stages. Six pts in the safety run-in received surufatinib at 250mg once daily as starting dose, in combination with a fixed dose of sintilimab (200 mg, d1, Q3W). If more than 1 DLT occurred in stage 1, the recommended dose of surufatinib for stage 2 (N=11) would de-escalate to 200 mg, otherwise would maintain as 250mg. The primary endpoint was ORR per RECIST v1.1. Secondary endpoints included DCR, PFS, OS and safety. If the ORR of part A was ≥ 30%, the trial would proceed to Part B (N=44), further evaluating this combination treatment in previously untreated advanced GC/GEJC. Results: At data cutoff on 8/10/2023, 16 pts (6 in safety run-in; 10 in dose expansion) were enrolled in part A. The median age was 56 years; 75% male; 69% with ECOG PS 1. Peritoneum (56%) was the most common metastatic site. No DLTs were observed in the safety run-in stage, so the recommended dose of surufatinib for dose expanding stage was 250 mg. Among the 16 pts evaluable for tumor response, 6 pts achieved PR, 4 pts achieved SD. The confirmed ORR and DCR per RECIST v1.1 was 40.0% (95% CI: 15.2- 64.6%) and 66.7% (95% CI: 38.4- 88.2%), respectively. Median DOR was not reached and 50% of responders were estimated to have a response duration ≥ 7.8 months. Median PFS and OS were not yet mature. Most TEAEs (≥ 40%) were grade 1-2, including hypoproteinemia (62.5%), hypertension (50%), proteinuria (50%), lymphocyte count decreased (43.8%). The most common grade ≥ 3 TEAEs reported by 5 pts (31.3%) were blood bilirubin increased (18.8%), hypertension, lymphocyte count decreased, and alanine transaminase elevation (6.3%, respectively). No treatment-related death was reported. Conclusions: The results showed surufatinib plus sintilimab acquired preliminary efficacy and manageable safety profile as the second-line treatment for advanced GC/GEJC. Part B is ongoing, and more data analysis will be reported further. Clinical trial information: NCT05235906 .

Acute myeloid leukemia with a novel AKAP9::PDGFRA fusion transformed from essential thrombocythemia: A case report and mini review

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy associated with various combinations of gene mutations, epigenetic abnormalities, and chromosome rearrangement-related gene fusions. Despite the significant degree of heterogeneity in its pathogenesis, many gene fusions and point mutations are recurrent in AML and have been employed in risk stratification over the last several decades. Gene fusions have long been recognized for understanding tumorigenesis and their proven roles in clinical diagnosis and targeted therapies. Advances in DNA sequencing technologies and computational biology have contributed significantly to the detection of known fusion genes as well as for the discovery of novel ones. Several recurring gene fusions in AML have been linked to prognosis, treatment response, and disease progression. In this report, we present a case with a long history of essential thrombocythemia and hallmark CALR mutation transforming to AML characterized by a previously unreported AKAP9::PDGFRA fusion gene. We propose mechanisms by which this fusion may contribute to the pathogenesis of AML and its potential as a molecular target for tyrosine kinase inhibitors.

Progress of non-small-cell lung cancer with ROS1 rearrangement.

ROS1 rearrangement is found in 0.9%-2.6% of people with non-small-cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs) target ROS1 and can block tumor growth and provide clinical benefits to patients. This review summarizes the current knowledge on ROS1 rearrangements in NSCLCs, including the mechanisms of ROS1 oncogenicity, epidemiology of ROS1-positive tumors, methods for detecting rearrangements, molecular characteristics, therapeutic agents, and mechanisms of drug resistance.

Evaluating Effectiveness and Safety of Flumatinib for Chronic Phase Chronic Myeloid Leukemia in (CML-CP) without Optimal Response (Warning, Failure) to Imatinib or/and Dasatinib

Introduction Flumatinib is a novel second-generation BCR-ABL1 targeted kinase inhibitors (TKI) with better efficacy and safety than first generation TKIs in first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). In the previous phase III study, newly diagnosed CML-CP patients receiving flumatinib achieved higher treatment responses and safety than imatinib. When it comes to the patients not achieving the optimal response to TKIs, flumatinib is potentially a better option, nonetheless the robust evidence is unavailable. This study reports the effectiveness and safety of switching to flumatinib in patients who have not achieved the optimal molecular response to imatinib or/and dasatinib. Methods This prospective, multicentre study enrolled Philadelphia Chromosome-Positive (Ph+) CML-CP patients aged ≥18 years and who failed to achieve the optimal response with imatinib or/and dasatinib between March 2021 to October 2022. All patients were treated with flumatinib 600 mg once a day and followed up to 24 months. The primary endpoint of the study was rate of major molecular response (MMR) at 12 months as defined by European LeukemiaNet 2020 recommendations (BCR-ABL1 transcript level ≤0.1% in peripheral blood on RT-PCR assay on International Scale [IS]) and safety was the secondary endpoint assessed with CTCAE 4.03 version. In addition, the optimal molecular response in either the warning or failure patients was evaluated at 3, 6 and 12 months respectively, after switching to flumatinib, and the difference in effectiveness was also analyzed from the available mutation subtypes. Results Overall, 94 patients with Ph+ CML-CP were included in the study. The median (range) age of the patients were 53 (19.0-84.0) years old and 61.7% of the patients were males. A total of 38 (40.4%) patients reported warning response from the previous TKIs whereas 56 (59.6%) of patients reported treatment failure to the previous TKIs. Seventy-seven (81.9%) patients have been treated with one TKI, either imatinib or dasatinib, and 17 (18.1%) have been treated with both prior to the study. Overall, 25 (54%) patients achieved MMR at 12-months follow-up compared to the baseline (Figure 1). The rates of optimal molecular response at 3- and 6-months were also improved compared to the baseline. The optimal molecular response was 76(88%) at 3 months, and 52(73%) at 6 months. Subgroup analysis with previous response to TKIs revealed that flumatinib improved the rates of optimal molecular response in both warning and failure groups. Switching to flumatinib from imatinib or/and dasatinib have been reported with an enhanced optimal molecular response in patients who experienced warning compared with those who had treatment failure after the TKIs with 38 (100%) vs 38 (79%), 29 (91%) vs 23 (59%) and 15 (68%) vs 10 (42%) at 3-, 6- and 12-months respectively (Figure 2). Similarly, the rates of optimal molecular response were improved in patients without mutations compared to those having mutations, 65 (90%) vs 9 (75%) at 3 months, 46 (77%) vs 5 (56%) at 6 months and 23 (59%) vs 1 (20%) at 12 months respectively. The most common adverse events (AEs) were diarrhea in 17 (18%), rash in 11 (12%), bloating in 11 (12%), and thrombocytopenia in 11 (12%) patients. Common hematological AEs of grade 3/4 were thrombocytopenia in 7 (7%), anemia in 3 (3%) and leucopenia in 1 (1%) patients. Conclusion For Ph+ CML-CP patients without the optimal molecular response in previous imatinib or/and dasatinib, flumatinib is a better alternative to the existing treatments. In addition, if the patients were switched to flumatinib earlier, the optimal response was achieved faster. The grade 3/4 AEs were mainly hematologically related, whereas the gastrointestinal events were mostly of grade 1-2.

Inhibition of PRKAA/AMPK (Ser485/491) phosphorylation by crizotinib induces cardiotoxicity via perturbing autophagosome-lysosome fusion

ABSTRACT Crizotinib, a small-molecule tyrosine kinase inhibitor targeting ALK, MET and ROS1, is the first-line drug for ALK-positive metastatic non-small cell lung cancer and is associated with severe, sometimes fatal, cases of cardiac failure, which increases the risk of mortality. However, the underlying mechanism remains unclear, which causes the lack of therapeutic strategy. We established in vitro and in vivo models for crizotinib-induced cardiotoxicity and found that crizotinib caused left ventricular dysfunction, myocardial injury and pathological remodeling in mice and induced cardiomyocyte apoptosis and mitochondrial injury. In addition, we found that crizotinib prevented the degradation of MET protein by interrupting autophagosome-lysosome fusion and silence of MET or re-activating macroautophagy/autophagy flux rescued the cardiomyocytes death and mitochondrial injury caused by crizotinib, suggesting that impaired autophagy activity is the key reason for crizotinib-induced cardiotoxicity. We further confirmed that recovering the phosphorylation of PRKAA/AMPK (Ser485/491) by metformin re-activated autophagy flux in cardiomyocytes and metformin rescued crizotinib-induced cardiomyocyte injury and cardiac complications. In summary, we revealed a novel mechanism for crizotinib-induced cardiotoxicity, wherein the crizotinib-impaired autophagy process causes cardiomyocyte death and cardiac injury by inhibiting the degradation of MET protein, demonstrated a new function of impeded autophagosome-lysosome fusion in drugs-induced cardiotoxicity, pointed out the essential role of the phosphorylation of PRKAA (Ser485/491) in autophagosome-lysosome fusion and confirmed metformin as a potential therapeutic strategy for crizotinib-induced cardiotoxicity. Abbreviations and Acronyms: AAV: adeno-associated virus; ACAC/ACC: acetyl-Co A carboxylase; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase; DAPI: 4ʹ6-diamidino-2-phenylindole; EF: ejection fraction; FOXO: forkhead box O; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HF: heart failure; HW: TL: ratio of heart weight to tibia length; IR: ischemia-reperfusion; KEGG: Kyoto encyclopedia of genes and genomes; LAMP2: lysosomal-associated membrane protein 2; LDH: lactate dehydrogenase; MCMs: mouse cardiomyocytes; MMP: mitochondrial membrane potential; mtDNA: mitochondrial DNA; MYH6: myosin, heavy peptide 6, cardiac muscle, alpha; MYH7: myosin, heavy peptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; PI3K: phosphoinositide 3-kinase; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; TKI: tyrosine kinase inhibitor; WGA: wheat germ agglutinin

EMP3 sustains oncogenic EGFR/CDK2 signaling by restricting receptor degradation in glioblastoma

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3’s stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.

Molecular pathology of non-small cell carcinoma.

Currently, lung cancer is treated by the highest number of therapeutic options and the benefits are based on multiple large-scale sequencing studies, translational research and new drug development, which has promoted our understanding of the molecular pathology of lung cancer. According to the driver alterations, different characteristics have been revealed, such as differences in ethnic prevalence, median age and alteration patterns. Consequently, beyond traditional chemoradiotherapy, molecular-targeted therapy and treatment with immune check-point inhibitors (ICI) also became available major therapeutic options. Interestingly, clinical results suggest that the recently established therapies target distinct lung cancer proportions, particularly between the EGFR/ALK and PD-1/PD-L1-positive subsets, e.g. the kinase inhibitors target driver mutation-positive tumours, whereas driver mutation-negative tumours respond to ICI treatment. These therapeutic efficacy-related differences might be explained by the molecular pathogenesis of lung cancer. Addictive driver mutations promote tumour formation with powerful transformation performance, resulting in a low tumour mutation burden, reduced immune surveillance, and subsequent poor response to ICIs. In contrast, regular tobacco smoke exposure repeatedly injures the proximal airway epithelium, leading to accumulated genetic alterations. In the latter pathway, overgrowth due to alteration and immunological exclusion against neoantigens is initially balanced. However, tumours could be generated from certain clones that outcompete immunological exclusion and outgrow the others. Consequently, this cancer type responds to immune check-point treatment. These pathogenic differences are explained well by the two-compartment model, focusing upon the anatomical and functional composition of distinct cellular components between the terminal respiratory unit and the air-conducting system.

Anti-proliferative activity of RIHMS-Qi-23 against MCF-7 breast cancer cell line is through inhibition of cell proliferation and senescence but not inhibition of targeted kinases

BackgroundBreast cancer is the most common malignancy globally, and is considered a major cause of cancer-related death. Tremendous effort is exerted to identify an optimal anticancer drug with limited side effects. The quinoline derivative RIMHS-Qi-23 had a wide-spectrum antiproliferative activity against various types of cancer cells.MethodsIn the current study, the effect of RIMHS-Qi-23 was tested on MCF-7 breast cancer cell line to evaluate its anticancer efficacy in comparison to the reference compound doxorubicin.ResultsOur data suggest an anti-proliferative effect of RIMHS-Qi-23 on the MCF-7 cell line with superior potency and selectivity compared to doxorubicin. Our mechanistic study suggested that the anti-proliferative effect of RIMHS-Qi-23 against MCF-7 cell line is not through targeted kinase inhibition but through other molecular machinery targeting cell proliferation and senescence such as cyclophlin A, p62, and LC3.ConclusionRIMHS-Qi-23 is exerting an anti-proliferative effect that is more potent and selective than doxorubicin.

Novel Mechanisms of Venetoclax Resistance in Acute Myeloid Leukemia Based on Genomic Rearrangements

Introduction: Venetoclax (VEN), a BCL-2 inhibitor, effectively induces apoptosis in acute myeloid leukemia (AML) cells. Resistance to VEN in the treatment of AML can occur through various mechanisms. Some of the identified resistance mechanisms include: expansion or gain of clones with activation of the receptor tyrosine kinase (RTK) pathway; disruption of TP53 function, a tumor suppressor gene known to play a critical role in cell cycle regulation and apoptosis; selection of AML populations with monocytic differentiation, possibly altering cellular characteristics and response to treatment; overexpression of the MCL1 gene, which encodes for a protein that can counteract the pro-apoptotic effects of venetoclax; switch to lipid metabolism, an adaptive response by leukemia cells that may confer resistance to venetoclax; mutations in BCL-2 and BAX genes, which could affect the activity of these key regulators of apoptosis. While these mechanisms have been extensively studied, the role of rearrangements, often combined with genomic events, has not been fully explored. The INTHEMA protocol (NCT04298892) aimed to identify new VEN resistance mechanisms in patients with relapsed/refractory AML treated with VEN in combination with hypomethylating agents and to provide addressable molecular pathways useful for restoring drug sensitivity or offer novel therapeutic opportunities. Methods: Informed consent was obtained from 21 consecutive patients. Samples were collected from these patients with relapse or refractory disease after VEN + hypomethylating agent treatment. TruSight RNA Pan-Cancer analysis was performed to investigate fusion genes (using capture technology covering 1385 genes), RNA expression, and expressed mutations for each sample. Results: Among the 21 patients, 17 (81%) showed at least one putative mechanism of VEN resistance (as illustrated in the figure). These mechanisms included co-occurring mutations and genomic rearrangements. RTK mutations were found in 14 patients (67%), with specific mutations observed in NRAS, PTPN11, CBL, FLT3, JAK2, cKIT, EPHB6, FGFR and PDGFRb. One patient had a BAX mutation (p.G67R; BH3 domain), three patients exhibited a switch to a monocytic phenotype, and two patients had marker chromosomes. Gene fusions were identified in eight patients (38%): in particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion ( BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with less defined roles ( PSPC1-ZMYM2, PER1-DDX5, ELL-SUGP2). Interestingly, most of the genes involved in traslocations, with a potential driver role in therapy resistance ( MECOM, ETV6, CCND2, BCL11B, NUP98) also showed high expression levels in patients harboring the traslocation. Unsupervised clustering of RNA expression data revealed two main patient groups. The first group (24%) demonstrated an “activating-like” signature, characterized by overexpression of CSF3R and IKZF, suggesting a potential target for tyrosine kinase inhibitors. The second group exhibited a “self-renewal like” signature, suggesting a possible benefit from histone deacetylase inhibitors, as previously published. Patients with gene fusions were predominantly clustered within the “self-renewal like” group, particularly in a subgroup (subgroup 4) showing overexpression of HOXA genes. Conclusions: Through deep transcriptomic characterization combined with conventional diagnostics, this analysis uncovered novel mechanisms of VEN resistance while confirming established ones. The distinct gene expression patterns may help tailor targeted therapies, with patients showing the “activating-like” signature potentially benefiting from tyrosine kinase inhibitors and those with the “self-renewal like” signature possibly responding well to histone deacetylase inhibitors. Furthermore, HOXA gene overexpression presents an exciting therapeutic opportunity for selected patients. Further research and clinical validation are needed to translate these findings into actionable clinical strategies.

P2.09-11 JIN-A04, Highly Effective and Brain-penetrant Tyrosine Kinase Inhibitor Targeting HER2 Exon20 Insertion Mutations in NSCLC

P2.09-11 JIN-A04, Highly Effective and Brain-penetrant Tyrosine Kinase Inhibitor Targeting HER2 Exon20 Insertion Mutations in NSCLC

Application of SNAP-tag-EGFR cell membrane chromatography model in screening antitumor active components of Silybum marianum (L.) Gaertn

The SNAP-tag-epidermal growth factor receptor (SNAP-tag-EGFR) cell membrane chromatography (CMC) model is a powerful tool for investigating ligand-receptor interactions and screening active ingredients in traditional Chinese medicine. Most tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptors. However, TKIs associated with significant side effects and drug resistance must be addressed immediately. Therefore, there is an urgent need to develop new TKIs with high efficiency and low toxicity. Because of its low toxicity and side effects, traditional Chinese medicine has been widely employed to treat various diseases, including cancer. Hence, this study aimed to use the SNAP-tag-EGFR/CMC-high-performance liquid chromatography-mass spectrometry (HPLC-MS) two-dimensional system model as the research tool to screen and identify potential EGFR antagonists from the Chinese medicine Silybum marianum (L.) Gaertn. The applicability of the system was verified using the positive control drug osimertinib. Four potential EGFR antagonists were screened from the Chinese medicine Silybum marianum (L.) Gaertn.. They were identified as silydianin, silychristin, silybin, and isosilybin. Additionally, their pharmacological activity was preliminarily verified using a CCK-8 assay. The kinetic parameters of the four active ingredients interacting with EGFR and their binding modes with EGFR were analyzed using nonlinear chromatography (NLC) and molecular docking. This study identified silydianin, silychristin, silybin, and isosilybin from Silybum marianum (L.) Gaertn. and verified their potential antitumor effects on EGFR.

Phase II study of apatinib plus exemestane in estrogen receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer

ABSTRACT Purpose Apatinib is a tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR)-2. This study was conducted to assess the efficacy and safety of apatinib combined with exemestane in patients with estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). Methods This single-center, single-arm phase II study enrolled patients with ER+/HER2- MBC progressed on previous letrozole or anastrozole. Stratified analysis was performed according to the number of chemotherapy regimens for metastatic disease. The primary endpoint was progression free survival (PFS). Secondary endpoints included objective response rate (ORR), disease control rate (DCR), clinical benefit rate (CBR), overall survival (OS) and toxicity. Patients received apatinib at a starting dose of 500 mg/d and exemestane 25 mg/d on days 1–28 of each 4-week cycle. Results Thirty patients were enrolled with median four prior anticancer therapies. Eighty percent of patients received chemotherapy for metastatic disease. The median PFS (mPFS) and OS were 5.6 (95%CI: 4.3–6.9) months and 15.7 (95% CI: 9.7–21.7) months, respectively. The ORR, DCR, and CBR were 21.4%, 71.4%, and 46.4%, respectively. Patients with 0–1 line chemotherapy for MBC showed a slightly longer mPFS compared to those with ≥2 lines chemotherapy (mPFS: 6.4 months vs 4.8 months, P = .090). Most of the AEs were grade 1/2. One patient (3.3%) who suffered bone marrow metastases experienced grade 4 thrombocytopenia, and 14 experienced grade 3 AEs. Fifty percent of patients were given reduced dose for apatinib. Conclusions Apatinib plus exemestane exhibited objective efficacy in patients with ER+/HER2- MBC who have failed multiple lines of treatment. The AEs of apatinib required close monitoring and most of patients were well tolerated.

MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling.

Despite the initial benefit from tyrosine kinase inhibitors (TKI) targeting oncogenic ALK and ROS1 gene fusions in non-small cell lung cancer, complete responses are rare and resistance ultimately emerges from residual tumor cells. Although several acquired resistance mechanisms have been reported at the time of disease progression, adaptative resistance mechanisms that contribute to residual diseases before the outgrowth of tumor cells with acquired resistance are less clear. For the patients who have progressed after TKI treatments, but do not demonstrate ALK/ROS1 kinase mutations, there is a lack of biomarkers to guide effective treatments. Herein, we found that phosphorylation of MIG6, encoded by the ERRFI1 gene, was downregulated by ALK/ROS1 inhibitors as were mRNA levels, thus potentiating EGFR activity to support cell survival as an adaptive resistance mechanism. MIG6 downregulation was sustained following chronic exposure to ALK/ROS1 inhibitors to support the establishment of acquired resistance. A higher ratio of EGFR to MIG6 expression was found in ALK TKI-treated and ALK TKI-resistant tumors and correlated with the poor responsiveness to ALK/ROS1 inhibition in patient-derived cell lines. Furthermore, we identified and validated a MIG6 EGFR-binding domain truncation mutation in mediating resistance to ROS1 inhibitors but sensitivity to EGFR inhibitors. A MIG6 deletion was also found in a patient after progressing to ROS1 inhibition. Collectively, this study identifies MIG6 as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors and suggests EGFR to MIG6 ratios and MIG6-damaging alterations as biomarkers to predict responsiveness to ALK/ROS1 and EGFR inhibitors.

Discovery of New Pyrrolo[2,3-d]pyrimidine Derivatives as Potential Multi-Targeted Kinase Inhibitors and Apoptosis Inducers.

In the pursuit of developing more potent and effective targeted kinase inhibitors (TKIs), a series of new compounds, specifically halogenated '(E)-4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N'-benzylidenebenzohydrazides', were successfully synthesized in three steps with high yields. Among these novel compounds, namely 5e, 5h, 5k, and 5l, promising cytotoxic effects were observed against four different cancer cell lines, with IC50 values ranging from 29 to 59 µM. Notably, compound 5k emerged as the most potent inhibitor, exhibiting significant activity against EGFR, Her2, VEGFR2, and CDK2 enzymes, with IC50 values ranging from 40 to 204 nM, comparable to the well-known TKI sunitinib (IC50 = 261 nM). Mechanistic investigations of compound 5k revealed its ability to induce cell cycle arrest and apoptosis in HepG2 cells, accompanied by a notable increase in proapoptotic proteins caspase-3 and Bax, as well as the downregulation of Bcl-2 activity. Furthermore, molecular docking studies indicated similar binding interactions between compound 5k and the four enzymes, as observed with sunitinib. These findings highlight the potential of compound 5k as a promising candidate for further development as a multi-targeted kinase inhibitor with enhanced potency.