CDK4/6 inhibition overcomes venetoclax resistance mechanisms with enhanced combination activity in acute myeloid leukemia

Abstract Cancer stem cells (CSCs) within glioblastoma (GBM) contribute to therapeutic resistance and tumor recurrence. Receptor tyrosine kinases (RTKs) such as VEGFR2, MET, and AXL are frequently dysregulated in GBM and implicated as GBM driver genes, making them attractive therapeutic targets. This study evaluates the efficacy of cabozantinib (XL184), a multi-kinase inhibitor targeting VEGFR2, MET, and AXL, in GBM CSCs and patient-derived xenograft (PDX), with a focus on uncovering resistance mechanisms and identifying opportunities for combination therapies. Nine genomically diverse GBM CSCs were treated with XL184 to assess drug sensitivity. Proteomics were used to evaluate changes in key signaling pathways. CSC-derived PDX from two proneural (HF2587, HF3016) and one classical (HF2927) GBM were treated with XL184 monotherapy. Tumor growth and survival were monitored. Immunohistochemical (IHC) analysis of CD31 expression to assess angiogenesis was performed with high-resolution microscopy. GBM CSCs exhibited a wide range of sensitivity to XL184 (IC50: 2μM - 34μM). Proteomics showed XL184-mediated inhibition of phospho-VEGFR2, AKT, and ERK, while sub-lethal XL184 doses upregulated phospho-MET and STAT3. In vivo, HF2587 (p=0.00022, n>18) and HF2927 (p=0.1847, n>12) xenografts responded to XL184 with reduced tumor growth and improved survival while HF3016 (p=0.061, n>16) was resistant to the monotherapy. HF2587 and HF2927 demonstrated a reduction in vessel density, measured by CD31 expression, compared to normal brain. The non-responsive HF3016 exhibited increased angiogenesis following therapy, a potential indicator of treatment resistance. These findings highlight the heterogeneity of XL184 response in GBM. Adaptive resistance through MET and downstream STAT3 activation supports combination therapies to overcome redundancy from pathway-specific negative feedback. Evidence of reduced angiogenesis imply that vasculature normalization is a key drug mechanism of action and support the theory of modulating the tumor microenvironment in glioma therapy.

Targeting AXL can effectively overcome c-Met-induced therapeutic resistance in renal cancer and promote tumor cell death through increased oxidative stress.

Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.

AXL in cardiovascular diseases: Pathophysiological insights and clinical perspectives.

Real-world efficacy of cabozantinib alone or in combination with ICIs in heavily pretreated hepatocellular carcinoma: potential role of AXL and MET expression: Cabozantinib ± ICIs in pretreated HCC patients.

Targeting AXL with zanzalintinib improves chemotherapy response in uterine serous cancer

BackgroundTumor-associated macrophages (TAMs) are key promoters of inflammatory breast cancer (IBC), the most aggressive form of breast cancer. The receptor tyrosine kinase AXL is highly expressed in various cancer types, including IBC, but its role in TAMs remains unexplored.MethodsWe examined the effects of AXL inhibitor TP-0903 on tumor growth and tumor microenvironment (TME) component M2 macrophages (CD206+) in IBC and triple-negative breast cancer mouse models using flow cytometry and immunohistochemical staining. Additionally, we knocked out AXL expression in human THP-1 monocytes and evaluated the effect of AXL signaling on immunosuppressive M2 macrophage polarization and IBC cell growth and migration. We then investigated the underlying mechanisms through RNA sequencing analysis. Last, we performed CIBERSORT deconvolution to analyze the association between AXL expression and tumor-infiltrating immune cell types in tumor samples from the Inflammatory Breast Cancer International Consortium.ResultsWe found that inhibiting the AXL pathway significantly reduced IBC tumor growth and decreased CD206+ macrophage populations within tumors. Mechanistically, our in vitro data showed that AXL promoted M2 macrophage polarization and enhanced the secretion of immunosuppressive chemokines, including CCL20, CCL26, and epiregulin, via the transcription factor STAT6 and thereby accelerated IBC cell growth and migration. RNA sequencing analysis further indicated that AXL signaling in immunosuppressive M2 macrophages regulated the expression of molecules and cytokines, contributing to an immunosuppressive TME in IBC. Moreover, high AXL expression was correlated with larger populations of immunosuppressive immune cells but smaller populations of immunoactive immune cells in tissues from patients with IBC.ConclusionsAXL signaling promotes IBC growth by inducing M2 macrophage polarization and driving the secretion of immunosuppressive molecules and cytokines via STAT6 signaling, thereby contributing to an immunosuppressive TME. Collectively, these findings highlight the potential of targeting AXL signaling as a novel therapeutic approach for IBC that warrants further investigation in clinical trials.

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME). Numerous studies have demonstrated a significant correlation between increased CAFs accumulation and poor outcomes in gastric cancer (GC) patients. However, no targeting agents against CAFs have been clinically applied in cancer treatments. AXL, a receptor tyrosine kinase, has emerged as a promising target to suppress cancer cell metastasis, yet AXL inhibitors have not been approved due to limited efficacy in clinical trials. However, the role of AXL upregulated in CAFs remains unexplored. In this study, we investigated the effect of AXL-positive CAFs on GC progression and explore their potential as therapeutic targets to enhance GC treatment efficacy. Methods: We analyzed a single cell RNA-seq (scRNA-seq) dataset of human GC tissues to identify the cell types expressing AXL. Human CAFs and mouse gastric fibroblasts (MGF) expressing AXL were treated with AXL inhibitors, such as CCB3233 and 9im. Additionally, AXL knockdown (KD) was performed in CAFs and MGFs, which were subsequently co-cultured with human GC cell lines (SNU668 or MKN-1) or mouse GC cell lines (ACKPY4113) to assess the effects of AXL+ fibroblasts on GC progression. The impact of AXL-inhibited fibroblasts on T-lymphocytes or macrophages was also evaluated. Furthermore, transcriptome analysis was conducted on AXL KD and control fibroblasts. Results: The analysis using scRNA-seq dataset revealed that AXL expression was predominantly upregulated in CAFs compared to other cell types. High expression and activation of AXL in fibroblasts were confirmed across various cell lines, including GC cell lines, fibroblasts and immune cells. AXL inhibitors, such as CCB3233 and 9im, effectively reduced AXL phosphorylation in human CAFs and MGFs. Co-culture experiments demonstrated that AXL-inhibited or KD fibroblasts significantly reduced the migration or invasion of GC cells compared to control fibroblasts. Moreover, while control CAFs and MGFs suppressed the activation of T- lymphocytes or macrophages, AXL-inactivated or KD fibroblasts were unable to inhibit immune cell activation. Transcriptome analysis of AXL KD or control fibroblasts revealed reduced expression of chemokines such as CXCL9 and CXCL13, which may explain the observed experimental results. Conclusions: Our findings highlight the crucial role of AXL-positive fibroblasts in promoting the aggressive phenotype of GC cells and suppressing immune cell activity in the GC TME. Targeting AXL in CAFs holds significant promise as a novel therapeutic strategy for GC treatment. Citation Format: Tuyen Thanh Tran, Yulim Lee, In-Hye Ham, Hur Hoon. AXL-positive cancer-associated fibroblasts enhance the metastatic potential of cancer cells and suppress tumor immunity in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2700.

Traditional cancer therapies often overlook the dynamic nature of cancer by utilizing single targets, considering only homogenous tumors, not considering the tumor microenvironment, and failing to overcome common issues such as induced and acquired resistance to therapy. The development of new therapies that address all these concerns remains a challenge in oncology today, and there is a need for single low-dose therapies that can specifically address heterogenous and complex populations. We propose a bispecific antibody targeting EGFR and AXL can address this gap, while considering cytotoxicity and avoiding off-target effects. EGFR is a receptor commonly overexpressed in many tumors and a known target in cancer therapy, however, many anti-EGFR treatments have detrimental off-target side effects for healthy cells. Additionally, resistance to anti-EGFR drugs is a common occurrence after prolonged treatment. One known mechanism of anti-EGFR resistance involves cross-talk between EGFR and the AXL receptor, a member of the TAM family of tyrosine kinase receptors which is also overexpressed in many tumors as well as in their immunosuppressive tumor microenvironments. AXL expression in oral cancers is known to mediate epithelial-mesenchymal transition through the PI3K pathway and has been shown to be associated with poor prognosis. New data from spatial tumor analysis of human patient derived xenografts shows huge diversity of expression of EGFR and AXL, both between tumors and within different cells of the same tumor where the expression levels of the two receptors appear to complement each other. This research includes the development and characterization of an IgG-like bispecific antibody targeting both EGFR and AXL receptors which we hypothesize can not only eliminate the immunosuppressive tumor microenvironment and tumors with heterogenous expression of the two targets, but could also prevent the common complication of acquired resistance as well as improve specificity and biodistribution of the therapy to reduce off-target effects.

Background: Head and neck cancer (HNC) evades immune responses by manipulating the tumor immune microenvironment (TIME). Tumor-bound Axl has been implicated in promoting an immunosuppressive TIME in HNC, though its precise role remains unclear. Understanding Axl's contribution to immune evasion in HNC could lead to the identification of new therapeutic targets; therapies directed at these targets could be combined with and thereby enhance immunotherapies. Results: Using Axl knockout (Axl KO) cell lines derived from the immunologically "cold" MOC2 mouse model, we found that Axl loss delayed tumor growth in immunocompetent mice. This was accompanied by reduced immunosuppressive cells, including MDSCs, Tregs, B cells, and neutrophils, and increased infiltration of cytotoxic CD8 T cells and NK cells. To identify the immune population(s) responsible for these changes, Axl KO tumors were implanted in immune-deficient mice. Axl KO tumor growth in athymic nude mice (which lack T cells) was unchanged, whereas tumor growth in NCG mice (which lack NK cells) was rescued, suggesting that NK cells mediate the Axl KO tumor growth delay. Further, Axl loss enhanced NK cell cytotoxicity in vitro and in vivo, and NK cell depletion reversed delayed Axl KO tumor growth. Mechanistically, Axl KO tumors showed decreased expression of CD73 and CCL2, which inhibit NK cells, and increased expression of CCL5 and CXCL10, which promote NK cell recruitment and activation. Conclusions: These novel findings suggest that tumor-bound Axl fosters an immunosuppressive TIME by inhibiting NK cell recruitment and function, thereby promoting tumor growth. Targeting Axl may enhance NK cell-mediated tumor killing and improve immunotherapy efficacy in HNC.

After failing primary and secondary hormonal therapy, castration-resistant and neuroendocrine prostate cancer metastatic to the bone is invariably lethal, although treatment with docetaxel and carboplatin can modestly improve survival. Therefore, agents targeting biologically relevant pathways in prostate cancer and potentially synergizing with docetaxel and carboplatin in inhibiting bone metastasis growth are urgently needed. Phosphorylated (activated) AXL expression in human prostate cancer bone metastases was assessed by IHC staining. We evaluated the effects of a novel soluble AXL signaling inhibitor, sAXL (batiraxcept or AVB-S6-500), on tumor growth and lung metastases in prostate cancer patient-derived xenograft models that were implanted intratibially. After injection of LuCaP cells into the tibiae, tumors were treated with batiraxcept and docetaxel or carboplatin alone or in combination, and tumor growth was monitored by serum prostate-specific antigen or bioluminescence. Tumor burden was quantified by human-specific Ku70 staining, and metastasis to the lungs was determined using qPCR. Transcriptomic profiling, Western blotting, and immunohistochemistry were performed to identify treatment-regulated gene and protein profile changes. High AXL phosphorylation in human prostate cancer bone metastases correlated with shortened survival. Batiraxcept alone or in combination with docetaxel or carboplatin significantly suppressed intratibial tumor growth and suppressed metastasis to the lungs through multiple mechanisms, including repression of cancer stemness genes (CD44, ALDH1A1, TACSTD2, and ATXN1) and the PI3K, JAK, MAPK, and E2F1/NUSAP1 signaling pathways. Our study provides a robust preclinical rationale and mechanisms of action for using batiraxcept as a single agent or in combination with docetaxel or carboplatin to treat lethal metastatic prostate cancer.

Hepatic ischemia-reperfusion (I/R) injury can cause poor prognosis of liver transplantation and hepatectomy, especially in patients with alcohol-associated liver disease (ALD). Apoptosis is closely related to different stages of liver injury, and the death of hepatocytes caused by endoplasmic reticulum (ER) and mitochondria homeostasis perturbation may be key to liver injury. The receptor tyrosine kinases AXL encoded by the gene axl, is a member of the TAM (TYRO3, AXL, and MERTK) family, which participates in various biological processes by binding to the ligand of growth arrest-specific protein 6 (Gas6). However, whether AXL is involved in apoptosis pathways, and the detailed mechanism in hepatic I/R injury remains unknown. In the present study, we found that total AXL is up-regulated while phosphorylated AXL (p-AXL, the active form of AXL) was down-regulated after I/R in human liver tissues from liver transplantation. Consistently, total AXL was found up-regulated while p-AXL was down-regulated during hepatic I/R injury in mice. Pretreatment with Gas6 increased p-AXL expression, reduced ER stress-associated cell apoptosis, alleviated liver damage, and restored ER and mitochondria ultrastructure during hepatic I/R in mice. Furthermore, the ALD model was established by chronic-plus-binge ethanol feeding to explore the role of AXL in I/R liver injury with ethanol-associated steatosis. We found that ALD mice had a lower p-AXL level and were more susceptible to hepatic I/R injury. Importantly, activated AXL ameliorated liver injury by inhibiting IRE1 and PERK pathway to reduce ER stress-associated apoptosis. In conclusion, activated AXL protects alcohol-associated steatotic liver against I/R injury by inhibiting ER stress and mitochondria-associated apoptosis, suggesting that targeting AXL serves as a potential strategy for liver I/R injury, particularly for marginal liver donors with alcohol-associated steatosis.

IntroductionThe incidence of chronic kidney disease (CKD) is increasing, in parallel with risk factors including obesity and diabetes mellitus. AXL plays a central role in CKD, providing a rationale to evaluate clinical AXL targeting agents.MethodsTo determine the efficacy and underlying molecular mechanisms of AXL inhibition in CKD, we employed a murine unilateral ureteral obstruction (UUO) model preventively treated with a selective AXL kinase inhibitor (bemcentinib) during disease progression. We isolated kidneys at an early (3 days) or late (15 days) timepoint and profiled the cell populations using mass cytometry.ResultsPreventive treatment with bemcentinib significantly attenuated fibrosis in the UUO model. The anti-fibrotic effect correlated with a decrease in mesangial cells and inhibition of innate immune cell infiltration, while the proportion of epithelial cells increased. We mapped AXL expression to a unique network of cells in the kidney: mesangial cells, pericytes, macrophages and dendritic cells.DiscussionWe propose that AXL targeting affects an important cellular interaction network underlying fibrotic progression. These results support the clinical application of AXL targeting agents to treat CKD.

160 (PB148): Targeting AXL kinase with Bemcentinib to enhance Type 1 IFN response and sensitize tumors to chemo-immunotherapy

Cabozantinib prevents the progression of metabolic dysfunction-associated steatohepatitis by inhibiting the activation of hepatic stellate cell and macrophage and attenuating angiogenic activity

A bispecific antibody targeting EGFR and AXL delays resistance to osimertinib

Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge in terms of diagnosis and treatment, with limited therapeutic options and a poor prognosis. This study explored the potential therapeutic role of NPS-1034, a kinase inhibitor targeting MET and AXL, in PDAC. The investigation included monotherapy with NPS-1034 and its combination with the commonly prescribed chemotherapy agents, fluorouracil and oxaliplatin. Our study revealed that NPS-1034 induces cell death and reduces the viability and clonogenicity of PDAC cells in a dose-dependent manner. Furthermore, NPS-1034 inhibits the migration of PDAC cells by suppressing MET/PI3K/AKT axis-induced epithelial-to-mesenchymal transition (EMT). The combination of NPS-1034 with fluorouracil or oxaliplatin demonstrated a synergistic effect, significantly reducing cell viability and inducing tumor cell apoptosis compared to monotherapies. Mechanistic insights provided by next-generation sequencing indicated that NPS-1034 modulates immune responses by inducing type I interferon and tumor necrosis factor production in PDAC cells. This suggests a broader role for NPS-1034 beyond MET and AXL inhibition, positioning it as a potential immunity modulator. Overall, these findings highlight the anticancer potential of NPS-1034 in PDAC treatment in vitro, both as a monotherapy and in combination with traditional chemotherapy, offering a promising avenue for further in vivo investigation before clinical exploration.

Melanoma is an aggressive, rapidly developing form of skin cancer that affects about 22 per 100,000 individuals. Treatment options for melanoma patients are limited and typically involve surgical excision of moles and chemotherapy. Survival has been improved in recent years through targeted small molecule inhibitors and antibody-based immunotherapies. However, the long-term side effects that arise from taking chemotherapies can negatively impact the lives of patients because they lack specificity and impact healthy cells along with the cancer cells. Antibody-drug conjugates are a promising new class of drugs for the treatment of melanoma. This review focuses on the development of antibody-drug conjugates for melanoma and discusses the existing clinical trials of antibody-drug conjugates and their use as a melanoma treatment. So far, the antibody-drug conjugates have struggled from efficacy problems, with modest effects at best, leading many to be discontinued for melanoma. At the same time, conjugates such as AMT-253, targeting melanoma cell adhesion molecule, andmecbotamab vedotin targeting AXL receptor tyrosine kinase, are among the most exciting for melanoma treatment in the future.

Abstract Adenoid cystic carcinoma (ACC) is a rare type of salivary gland cancer. Despite the many advances made in helping understand the disease behavior and characteristics, the prevailing challenge in managing patients with ACC is the lack of clinically approved systemic therapy drugs to successfully treat those with advanced recurrent or metastatic disease. There are multiple promising candidate agents being evaluated preclinically and in early phase clinical trials, yet an effective drug with consistent overall response remains to be identified. AXL is a member of the TAM family of tyrosine kinase receptors, and its overexpression and activation is generally associated with poor prognosis and chemoresistance in many cancer types. Previous reports show AXL is significantly upregulated in a subset of ACC tumors. Here, we evaluate the tumor growth inhibition effects of a newly developed antibody-drug-conjugate targeting AXL (ADCT-601) in a xenograft ACC cell line model. MDA-ACC-01 cells express high levels of cell surface AXL as confirmed by biochemical analysis. The tumor growth inhibition of MDA-ACC-01 cell xenograft was tested in a single-dose drug administration study. Immunodeficient mice bearing ACC tumors were either injected with vehicle (n=7) or with ADCT-601 at 0.5 (n=9) or 1.0 mg/kg (n=7). ADCT-601 treatment with either 0.5 mg/kg or 1.0 mg/kg resulted in complete response (CR) for all tumor implants. To reaffirm the drug’s efficacy, we challenged the vehicle-treated tumor, prior to exceeding the tumor volume endpoint, with a single-dose of ADCT-601 at 1.0 mg/kg. Strikingly, by day 6 post-treatment, tumor volume began to shrink and eventually resulted in CR except in all but one case. These results demonstrate ADCT-601 is an effective drug with significant anti-tumor activity in our preclinical models and is worthy of additional investigation to determine its translational potential. Citation Format: Joseph O. Humtsoe, Anita Pothukuchi, Hyunseok Kang, Patrick Ha. Preclinical anti-tumorigenic evaluation of AXL targeting antibody-drug-conjugate in an adenoid cystic carcinoma cell line xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB022.