Targeting AXL Research Articles

Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia

AbstractThe abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

Abstract LT021: Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells

Abstract Breast cancer-related deaths are predominantly associated to complications arising from metastases. Hypoxia is an important biological process that fuels metastasis and contributes to deregulate the tumor microenvironment (TME) of solid tumors. Hypoxia also promotes resistance to therapies by a plethora of mechanisms including enhancing tumoral angiogenesis, metabolic reprogramming, epithelial-to-mesenchymal transition (EMT) and immune evasion. The expression of the receptor tyrosine kinase AXL, a hypoxia-induced gene, is associated with poor clinical prognosis and metastasis in various cancers, including breast cancer. Here, we hypothesize that genetic or pharmacological interference of AXL may alter hypoxic responses and associated modulation of the TME offering an ideal setting for immunotherapy. In a HER2 mouse model of breast cancer (MMTV-NIC), we found that the mammary gland epithelial cells specific deletion of AXL led to a reduction of lung metastases and improved the TME by reducing the hypoxic response in tumor cells. When breast cancer cells were subjected to hypoxic conditions, we found that AXL inhibition reduced the levels of the hypoxia transcription factor HIF-1a which resulted in an altered hypoxic response. Specifically, AXL deletion led to a reduction of hypoxia-induced EMT and invasion, and a reduction of the secretion of key cytokines for macrophages recruitment and polarization. We demonstrate in vivo that AXL pharmacological inhibition in the MMTV-NIC breast cancer model generated an anti-inflammatory TME that enhanced an anti-PD-1 immune checkpoint blockade response and decreased the metastatic burden. Collectively, these results suggest that targeting AXL could be a powerful approach to improve immunotherapy response by generating an anti-tumoral microenvironment therefore limiting the metastatic burden of breast cancer. Citation Format: Marie-Anne Goyette, Jean-Philippe Gratton, Jean-François Côté. Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT021.

Cooperative Targeting of Immunotherapy-Resistant Melanoma and Lung Cancer by an AXL-Targeting Antibody-Drug Conjugate and Immune Checkpoint Blockade.

Although immune checkpoint blockade (ICB) has shown remarkable clinical benefit in a subset of patients with melanoma and lung cancer, most patients experience no durable benefit. The receptor tyrosine kinase AXL is commonly implicated in therapy resistance and may serve as a marker for therapy-refractory tumors, for example in melanoma, as we previously demonstrated. Here, we show that enapotamab vedotin (EnaV), an antibody-drug conjugate targeting AXL, effectively targets tumors that display insensitivity to immunotherapy or tumor-specific T cells in several melanoma and lung cancer models. In addition to its direct tumor cell killing activity, EnaV treatment induced an inflammatory response and immunogenic cell death in tumor cells and promoted the induction of a memory-like phenotype in cytotoxic T cells. Combining EnaV with tumor-specific T cells proved superior to either treatment alone in models of melanoma and lung cancer and induced ICB benefit in models otherwise insensitive to anti-PD-1 treatment. Our findings indicate that targeting AXL-expressing, immunotherapy-resistant tumors with EnaV causes an immune-stimulating tumor microenvironment and enhances sensitivity to ICB, warranting further investigation of this treatment combination. SIGNIFICANCE: These findings show that targeting AXL-positive tumor fractions with an antibody-drug conjugate enhances antitumor immunity in several humanized tumor models of melanoma and lung cancer.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Targeting AXL Kinase Sensitizes Acute Myeloid Leukemia Stem and Progenitor Cells to Venetoclax Treatment

Acute myeloid leukemia (AML) is a heterogeneous haematological cancer characterized phenotypically by the rapid clonal growth of myeloid cells and an accumulation of blasts in the peripheral blood and bone marrow of patients. Despite the major progress that has been made in categorizing different genetic and molecular AML subgroups, therapies and long-term patient outcomes have not changed significantly over the past four decades. Recently, venetoclax (ABT-199), a BH3-mimetic and selective BCL-2 inhibitor, was approved for the treatment of older patients with AML. However, the limited efficacy, drug resistance in complex karyotype AML and disease progression on venetoclax as well as the inherent resistance of leukemic stem cells (LSCs) to therapy pose significant clinical challenges, warranting identification of novel targets and improved treatment strategies. One candidate target is AXL, a member of the TYRO3/AXL/MER (TAM) family of receptor tyrosine kinases. AXL and its ligand growth arrest-specific gene 6 (GAS6) are elevated in AML patients and LSCs, and associated with poor prognosis. To test whether targeting of the AXL/GAS6 pathway is a feasible treatment strategy for AML, in particular to eradicate LSCs, we developed SLC-391, a novel, potent and selective AXL inhibitor. In vitro and in vivo evaluations of the pharmaceutical properties of SLC-391 indicated reasonable solubility, excellent metabolic stability as well as desirable bioavailability in mice and rats. In silico molecular docking analysis showed that SLC391 can adopt a conformation with surface and charge complementary to the active site of the AXL kinase, potentially engaging in hydrophobic ring-mediated interactions. Further, cell-based studies discovered that SLC-391 targets AML cells with high AXL/GAS6 expression, particularly MLL+ AML cells, and synergizes with venetoclax in cell viability and apoptosis assays (CI<0.6). In addition, simultaneous AXL and BCL-2 inhibition reduced the clonal short- and long-term growth of primitive AML patient cells in CFC re-plating and LTC-IC assays compared to single or control treatments (20-95% inhibition). Moreover, a combination of AXL inhibition and venetoclax treatment was able to target LSCs and AML blasts in two different preclinical patient-derived xenotransplantation (PDX) models, extending the mean survival of these mice by 14-30 days compared to single agents (P<0.025). Mechanistically, single-cell RNA-sequencing and functional validation studies revealed that AXL inhibition perturbs oxidative metabolism, and differentially targets signaling pathways to synergize with venetoclax in leukemic cell killing. Importantly, the combination of AXL inhibition plus venetoclax treatment was not toxic to normal BM cells from healthy donors. Hence, our findings identify a promising, improved and specific treatment strategy for AML, particularly patients with high AXL/GAS6 expression. Disclosures No relevant conflicts of interest to declare.

IJU this issue

This issue contains 15 Original Articles, three of which are excellent prospective clinical trials, and one Urological Note. Cabozantinib is a multiple receptor tyrosine kinase inhibitor targeting MET (c-MET), VEGFR2, RET, AXL, KIT and TIE-2. In the phase III, randomized, open-label METEOR study, cabozantinib was associated with a significant progression-free survival benefit and a greater objective response rate versus everolimus in patients with metastatic renal cell carcinoma who had progressed after VEGFR-TKI. Tomita et al. (Niigata, Japan) carried out a bridging study to METEOR to evaluate the efficacy and safety of cabozantinib in Japanese patients. This phase II, open-label, single-arm study showed that cabozantinib provided clinical benefits in Japanese patients with renal cell carcinoma, with similar efficacy and safety to the non-Japanese population. Medical lasers are increasingly used to treat benign prostatic hyperplasia. Shoji et al. (Isehara, Japan) carried out a randomized prospective study to compare functional outcomes and complications over a period of 12 months in benign prostatic hyperplasia treated with thulium laser enucleation of the prostate or bipolar transurethral resection of the prostate. This study showed that thulium laser enucleation of the prostate is superior to bipolar transurethral resection of the prostate in resected volume, resected rates per prostate volume and transition zone volume, hemostasis, catheterization and hospitalization time, and decrease in prostate-specific antigen. However, excessive laser irradiation time in thulium laser enucleation of the prostate has the possibility to delay improvement of overactive bladder symptoms and decrease sexual function. Loose seeds and stranded seeds are available for low-dose rate brachytherapy. Each method has its own advantages and disadvantages, such as flexibility and fixation. Tanaka et al. (Kashihara, Japan) carried out a randomized controlled trial to compare the post-implant dosimetric parameters between the conventional method (using only the loose seeds) and the hybrid method. This study showed that the hybrid method of low-dose rate brachytherapy can achieve a higher coverage of the periprostatic region compared with the conventional method, while maintaining an acceptable level of urethral and rectal doses. The intravesical recurrence rate after transurethral resection of bladder tumor carried out for non-muscle-invasive bladder cancer (NMIBC) is high, and some risk factors for recurrence have been identified. Sazuka et al. (Chiba, Japan) evaluated the risk of the residual urine volume and preoperative pyuria in intravesical recurrence after transurethral resection of bladder tumor among NMIBC patients. They found that the presence of residual urine and pyuria raised the risk of intravesical recurrence after transurethral resection of bladder tumor for NMIBC. Furthermore, especially in men, the recurrence-free survival was significantly higher in patients without than with residual urine by propensity score matching analysis. Iwasawa et al. (Tokyo, Japan) investigated the relationship between fluid intake and tumor recurrence in NMIBC patients by using urine-specific gravity as an indicator of daily fluid intake. They found that high urine-specific gravity was associated with tumor recurrence, especially in patients not receiving adjuvant bacillus Calmette–Guérin therapy. This showed that increasing fluid intake might prevent recurrence in relatively low-risk NMIBC patients. Testicular microlithiasis (TM) is a relatively rare condition occurring in the testicular parenchyma that is characterized by multiple bright and small foci without acoustic shadow limited to the testicle on scrotal ultrasonography. TM is a characteristic of testicular dysgenesis syndrome and was shown to be associated with infertility. Hiramatsu et al. (Tokyo, Japan) evaluated the correlation between the degree of severity in TM and several factors. This study showed the prevalence of TM in normal healthy Japanese men who are trying for first-time pregnancy. This also showed that men with TM might have poor semen findings, and that men with severe TM might have even worse semen parameters. None declared.

Abstract 3052: Targeting AXL in PIK3R2-amplified ovarian cancer

Abstract The p85 regulatory subunit and its associated p110 catalytic subunit are components of the class IA PI3K that mediates the downstream signal of receptor tyrosine kinases. PIK3R2 (encoding p85β, one of the p85 isoforms) is commonly amplified in ovarian cancer patients. Importantly, high PIK3R2 expression significantly correlated with worse patient survival. Here, we showed that p85β promoted tumorigenic properties of ovarian cancer cells. The protein expression of a receptor tyrosine kinase AXL was positively regulated by p85β. AXL is a member of the TAM receptor family. The regulation was specific to AXL because the other two members (Tyro3 and MERTK) were not altered by p85β. Drug sensitivity assays suggested that inhibition of AXL reduced oncogenic activities of p85β in vitro and in vivo. Together, we propose p85β as a potential therapeutic target in ovarian cancer and therapeutic blockade of AXL signaling may be an effective strategy for ovarian cancer with PIK3R2 amplification. Citation Format: Ling Rao, Victor C. Mak, Yuan Zhou, Dong Zhang, Xinran Li, Yiling Lu, Gordon B. Mills, Lydia W. Cheung. Targeting AXL in PIK3R2-amplified ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3052.

Role of AXL in metastatic melanoma and impact of TP-0903 as a novel therapeutic option for melanoma brain metastasis.

e22021 Background: Melanoma brain metastases (MBM) are common with a median overall survival of 4-5 months. Although immunotherapies have improved clinical outcomes and have doubled overall survival in MBM, there is a high incidence rate of relapse caused by drug resistance. AXL, a receptor tyrosine kinase (RTK), is associated with drug resistance and metastasis in many cancers. The activation of AXL via trans-phosphorylation regulates multiple signaling pathways that induce tumor survival, metastasis, drug resistance, and epithelial-to-mesenchymal transition (EMT). In MBM, AXL is upregulated and associated with disease progression, promoting cell invasion and migration. This suggests that targeting AXL can be a novel strategy to overcome treatment-related resistance in MBM. TP-0903, an investigational small molecule inhibitor of AXL, has shown efficacy in reversing the mesenchymal phenotype and re-sensitizing resistant cancer cells to targeted therapies in heme malignancies, pancreatic, and breast cancer. We aim to investigate the efficacy of TP-0903 in MBM. Methods: The Cancer Genome Atlas (TCGA) data was utilized to investigate the signaling pathways downstream of AXL that are upregulated in advanced melanoma. Nine signaling molecules including AKT1, mTOR, and PAK4 were analyzed to identify any correlation between gene expression levels and overall survival. Four metastatic melanoma cell lines were used to evaluate the effect of TP-0903 on cell viability and active AXL downregulation was assessed in vitro through MTS cell viability assays and Immunoblotting. Wound closure assays were executed to understand the functional consequences of AXL downregulation. Results: In all nine genes, high expression levels confer poor survival probability. Cell viability assays of four malignant melanoma cell lines showed that TP-0903 treatment resulted in IC50 values ranging from 32 – 692 nM. Western blot analysis indicated that TP-0903 reduced the levels of phosphorylated AXL in malignant melanoma cell lines. In addition, increasing TP-0903 concentrations reduced the rate of cell migration in these malignant melanoma cell lines. Conclusions: AXL plays a role in EMT, treatment resistance, and metastasis in MBM, resulting in poor survival. Our findings suggest TP-0903 is effective in reducing cell migration, inhibit metastasis, and can be a potential therapeutic option in MBM.

We couldn’t resist comparing central with local RECIST1.1 and with Choi assessment: Exploratory analysis of tumor imaging in EORTC STBSG Phase 2 trial 1317 “CaboGIST.

e23563 Background: Gastrointestinal stromal tumor (GIST) is commonly driven by activating mutations in KIT or PDGFRA. Advanced GIST is treated with tyrosine kinase inhibitors (TKIs) but develops resistance over time. EORTC 1317 assessed the safety and activity of cabozantinib, a multi-TKI targeting KIT, MET, AXL and proangiogenic pathways, in GIST patients who had progressed on imatinib and sunitinib. The efficacy analysis of the trial, which met its primary endpoint, was based on local assessment of RECIST1.1 response. RECIST neglects myxoid degeneration, necrosis and vascular remodeling induced by TKIs without major volumetric changes. Density changes on CT scans can predict clinical benefit and can be assessed by Choi criteria. Methods: We describe results of a post hoc exploratory analysis of CT scans performed centrally using RECIST 1.1 and Choi criteria. Results: Week 12 scans were available and evaluable by central review in 43 pts, Choi in 42 cases. Comparisons between local and central RECIST1.1 outcome revealed discrepancies in 17/43 evaluable cases (39.5%). When comparing Choi with local and central RECIST1.1 at week 12, discrepancies were observed in 27/42 (64.3%) and 21/42 (50%) evaluable cases, respectively. In summary, 70% of evaluable patients were progression-free and alive at week 12 based on local assessment, 86% and 83% according to central RECIST1.1 and Choi criteria, respectively. The main difference was the rate of objective response with cabozantinib in week 12: 5 PR (12%) with local RECIST1.1, 3 PR (7%) with central assessment, and 21 PR (50%) with Choi criteria. Conclusions: RECIST1.1 remains an unsatisfactory tool for response assessment in GIST, illustrated by the high inter-rater variability of response outcome comparing local versus central analysis. RECIST1.1 clearly underestimates the anti-tumor activity of TKIs in GIST. Cabozantinib did not only meet the primary endpoint of this trial when applying RECIST1.1 per protocol, but achieved objective responses in 50% of evaluable patients in week 12 when using Choi criteria. [Table: see text]

A phase II trial of cabozantinib and erlotinib for patients with EGFR and c-Met co-expressing metastatic pancreatic adenocarcinoma (PDAC).

e16764 Background: Both EGFR and the c-MET receptors are overexpressed in a majority of PDACs. Inhibition of both receptors simultaneously may be required for anti-tumor activity. Erlotinib, an EGFR inhibitor, has modest activity in metastatic PDAC and is approved by the FDA in combination with gemcitabine. Cabozantinib is a tyrosine kinase inhibitor targeting AXL, FLT-3, KIT, MER, MET, RET, ROS1, TIE-2, TRKB, TYRO3, and VEGFR-1, -2, and -3. Preclinical data suggests that the addition of cabozantinib to erlotinib leads to tumor shrinkage and improvement in survival in a KPC PDAC mouse model compared to gemcitabine alone. This phase II study tests this hypothesis in patients with metastatic PDAC that co-express c-MET and EGFR. Methods: Key eligibility includes patients (pts) with metastatic PDAC with EGFR and c-MET overexpression (as determined by centrally tested IHC of 2+ or greater) that have progression on one prior chemotherapy regimen. Patients were treated with cabozantinib (40mg daily) and erlotinib (100mg daily) continuously. This dosing is based on previous combination data in NSCLC. This is a single arm two-stage phase II study with a primary endpoint of overall response rate. Secondary endpoints include of PFS, DCR and OS. Results: From October 2017 to October 2019, 43 pts were screened with 7 pts (median age 62 [range 51-76)] enrolled and treated on study. Pts had a median of 1 line of prior systemic chemotherapy. Most common reason for screen failure was due to lack of co-expression of c-MET and EGFR. EGFR IHC expression was +2 in 4 pts, +3 in 3 pts; c-MET IHC expression was +2 in 5 pts and +3 in 2 pts. Most common any-grade adverse events attributable to cabozantinib and erlotinib include: diarrhea (71%), AST increase (43%), fatigue (43%), nausea (43%), and rash (43%). Only one grade 3 event of fatigue occurred. All pts had clinical and/or radiographic progression within 1-2 months after initiating study therapy. Conclusions: The combination of cabozantinib and erlotinib was well tolerated with manageable toxicity. Due to lack of clinical responses, this study has been terminated due to futility. Clinical trial information: NCT03213626 .

Targeting AXL and the DNA Damage Response Pathway as a Novel Therapeutic Strategy in Melanoma.

Receptor tyrosine kinase AXL is found upregulated in various types of cancer, including melanoma, and correlates with an aggressive cancer phenotype, inducing cell proliferation and epithelial-to-mesenchymal transition. In addition, AXL has recently been linked to chemotherapy resistance, and inhibition of AXL is found to increase DNA damage and reduce expression of DNA repair proteins. In light of this, we aimed to investigate whether targeting AXL together with DNA damage response proteins would be therapeutically beneficial. Using melanoma cell lines, we observed that combined reduction of AXL and CHK1/CHK2 signaling decreased proliferation, deregulated cell-cycle progression, increased apoptosis, and reduced expression of DNA damage response proteins. Enhanced therapeutic effect of combined treatment, as compared with mono-treatment, was further observed in a patient-derived xenograft model and, of particular interest, when applying a three-dimensional ex vivo spheroid drug sensitivity assay on tumor cells harvested directly from 27 patients with melanoma lymph node metastases. Together, these results indicate that targeting AXL together with the DNA damage response pathway could be a promising treatment strategy in melanoma, and that further investigations in patient groups lacking treatment alternatives should be pursued.

A phase II study investigating cabozantinib in patients with refractory metastatic colorectal cancer (AGICC 17CRC01).

103 Background: Therapeutic resistance to antiangiogenics in metastatic colorectal cancer (mCRC) inevitably develops via multiple mechanisms including upregulation of the MET kinase pathway. Cabozantinib, an oral multityrosine kinase inhibitor targeting MET, AXL, and VEGFR, demonstrated significant anti-tumor activity in CRC xenograft and cell line models. Methods: A single-arm, two-stage phase II study was conducted at 7 AGICC centers nationwide. 44 patients (pts) with mCRC who had progressed on or were intolerant of standard of care agents were treated with cabozantinib 60 mg daily in q3 wk cycles. The primary endpoint was 12-wk PFS rate. Based on the control arm of phase III CORRECT study, the Kaplan-Meier 12-wk PFS rate estimate was 13% and served as the null hypothesis. This study was powered at 0.906 to detect the alternative hypothesis of 12-wk PFS rate of 33% with a type I error rate of 0.044. Secondary endpoints were safety, RR, OS, and retrospective analysis of PFS and RR based on RAS, BRAF, and PIK3CA mutation status. Results: 44 pts were enrolled and 34 pts were response-evaluable as having undergone at least the first 6-wk restaging scan. 10 pts discontinued treatment prior to the first 6-wk scan due to clinical disease progression. Median number of cycles was 4 and median follow-up was 2.5 months. As of data-cutoff 8/23/2019, 55 Grade 3/4 AEs were reported with the most common being hypertension, fatigue, diarrhea, pain, HFS, nausea, vomiting, and proteinuria. 32 SAEs occurred in 18 pts. 5 Grade 5 AEs were reported: disease progression (3), disseminated intravascular coagulopathy, and bowel perforation. 15 pts (34%) achieved ≥ 12-wk PFS and 8 patients remain on treatment. Best response was 1 PR and 31 SD with a DCR at 6 wks of 72.7%. Of the pts who achieved ≥ 12-wk PFS, 12 had left-sided primary tumors, 5 had a RAS mutation, 1 had a PIK3CA mutation, and all pts were BRAF WT and MSI stable. Conclusions: Cabozantinib was deemed safe and demonstrates encouraging efficacy in a heavily pretreated mCRC pt population. These results support further investigation of cabozantinib in mCRC. Clinical trial information: NCT03542877.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

Introduction:Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC.Methods:We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC.Results:We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018).Conclusion:Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling.The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor.Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.

Targeting AXL and RAGE to prevent geminin overexpression-induced triple-negative breast cancer metastasis

Dissemination of metastatic precursors from primaries is the primary reason for patient death. Dissemination encompasses tumor cells invasion of stroma, followed by intravasation through the endothelium barrier into the bloodstream. Here, we describe how geminin-overexpressing tumor cells acquire dissemination ability. Acetylated HMGB1 (Ac-HMGB1) secreted by geminin-overexpressing cells activates RAGE and CXCR4 expression on mesenchymal stem cells (MSCs) located in tumor stroma. Through secreting CXCL12, geminin-overexpressing cells recruit these CXCR4+-MSCs into the tumor. Within the tumor, MSCs differentiate into S100A4-secreting cancer-associated fibroblasts (CAFs). S100A4, in a reciprocal manner, activates geminin-overexpressing cells to secrete CCL2 that recruits M0-macrophages from the stroma into the tumor. Within the tumor, CCL2 polarizes M0-macrophages into Gas6-secreting M2-tumor-associated macrophages (M2-TAMs). In concert, geminin-overexpression, S100A4/RAGE and Gas6/AXL signaling promote the invasive and intravasation abilities in geminin-overexpressing cells through exacerbating their stemness and epithelial-to-mesenchymal phenotypes and enhancing expression and functional interaction of CD151 and α3β1-integrin in geminin-overexpressing cells. Tumors formed following injection of geminin-overexpressing cells admixed with MSCs/CAFs grew faster, metastasized earlier, especially to lungs, and were extremely sensitive to anti-c-Abl, anti-RAGE, and anti-AXL drugs. These data support an intrinsic ability in geminin-overexpressing tumor cells to promote their metastatic potential through recruitment and bi-directional interactions with MSCs/CAFs and M2-TAMs.

Abstract C016: Oxidative phosphorylation is a metabolic vulnerability in chemotherapy resistant triple negative breast cancer

Abstract There is a pressing need to identify improved therapies for triple negative breast cancers (TNBC) resistant to standard chemotherapy. To identify potential molecular targets, we performed RNA sequencing of pre-treatment biopsies from 43 patients with operable TNBC who received neoadjuvant anthracycline and taxane-based chemotherapy. Ingenuity pathway analysis demonstrated that the top canonical pathway associated with higher likelihood of recurrence was higher expression of oxidative phosphorylation (OXPHOS) signature. We therefore sought to determine the efficacy of IACS-10759, a potent inhibitor of OXPHOS, in 10 TNBC patient-derived xenografts (PDX), 8 generated from chemotherapy-resistant tumors. Partial response was observed in one PDX model and prolonged disease stabilization in 5 of 10 PDXs. PDXs with higher expression of protein coding mitochondrial genes were more sensitive to IACS-10759. AXL overexpression was associated with intrinsic and acquired IACS-10759 resistance. The combination of cabozantinib, a multi-kinase inhibitor targeting AXL, with IACS-10759 significantly improved responses in TNBC PDXs. In contrast, selective AXL inhibitor BGB324 or knockdown of AXL did not enhance IACS-10759 sensitivity. In addition, an in vivo synthetic lethality screen identified CDK4, PARP1 and PARP2 as potential combination targets for IACS-10759. Palbociclib as well as talazoparib enhanced growth inhibitory effect of OXPHOS inhibition in vitro and in vivo. Our data suggests that OXPHOS is a promising target in chemoresistant TNBC. IACS-10759 is currently in Phase 1 testing, including TNBC. Further work is needed to determine the optimal biomarker-driven combination partners. Citation Format: Kurt Evans, Stacy Moulder, Erkan Yuca, Stephen Scott, Natalia Paez Arango, Maryam Shariati, Christopher P Vellano, Turcin Saridogan, Xiaofeng Zheng, Ana Maria Gonzalez-Angulo, Ming Zhao, Xiaoping Su, Coya Tapia, Ken Chen, Argun Akcakanat, Charles M Perou, Bora Lim, Debu Tripathy, Timothy A Yap, Maria E Di Francesco, Giulio Draetta, Philip Jones, Joe Marszalek, Funda Meric-Bernstam. Oxidative phosphorylation is a metabolic vulnerability in chemotherapy resistant triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C016. doi:10.1158/1535-7163.TARG-19-C016

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)- and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

Targeted agents for second-line treatment of advanced hepatocellular carcinoma.

Over the past ten years, sorafenib, a multikinase inhibitor, has been the standard of care for patients with unresectable hepatocellular carcinoma (HCC) and well-preserved liver function. Recently, lenvatinib, a different multikinase inhibitor, was shown to be non-inferior to sorafenib, in terms of survival, while all other agents previously tested failed to prove non-inferiority (or superiority) when compared to sorafenib. Similarly, in the second-line setting, most investigational drugs failed to provide better survival outcomes than placebo. However, in the last 2 years three positive phase III trials have been published in this setting. The RESORCE trial, a phase III study evaluating regorafenib in HCC patients who experienced disease progression after first-line treatment with sorafenib, showed better outcomes with regorafenib compared to placebo. More recently, the phase III CELESTIAL trial demonstrated the superiority of cabozantinib, a multikinase inhibitor targeting vascular endothelial growth factor receptor, MET, and AXL, vs placebo in the second- and third-line setting in patients progressing on or intolerant to sorafenib. The survival benefits of a sustained anti-angiogenic inhibition were demonstrated also with ramucirumab in the phase III REACH-2 trial in patients previously treated with sorafenib and who had high baseline alpha-fetoprotein levels. Overall, the adverse events reported in these trials were in line with the known safety profiles of the tested agents. After nearly a decade of a certain degree of stagnation, we are now witnessing a period of novel therapeutic advances with multikinase inhibitors and monoclonal antibodies that will likely change the treatment scenario of HCC.

Liquid biopsy-based single-cell metabolic phenotyping of lung cancer patients for informative diagnostics

Accurate prediction of chemo- or targeted therapy responses for patients with similar driver oncogenes through a simple and least-invasive assay represents an unmet need in the clinical diagnosis of non-small cell lung cancer. Using a single-cell on-chip metabolic cytometry and fluorescent metabolic probes, we show metabolic phenotyping on the rare disseminated tumor cells in pleural effusions across a panel of 32 lung adenocarcinoma patients. Our results reveal extensive metabolic heterogeneity of tumor cells that differentially engage in glycolysis and mitochondrial oxidation. The cell number ratio of the two metabolic phenotypes is found to be predictive for patient therapy response, physiological performance, and survival. Transcriptome analysis reveals that the glycolytic phenotype is associated with mesenchymal-like cell state with elevated expression of the resistant-leading receptor tyrosine kinase AXL and immune checkpoint ligands. Drug targeting AXL induces a significant cell killing in the glycolytic cells without affecting the cells with active mitochondrial oxidation.

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells

AbstractThe era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.