ABSTRACT
Doxorubicin (doxo) remains the standard of care for patients with advanced soft tissue sarcoma (STS), even though response rates to doxo are only around 14% to 18%. We evaluated enapotamab vedotin (EnaV), an AXL-specific antibody-drug conjugate (ADC), in a panel of STS patient-derived xenografts (PDX). Eight models representing multiple STS subtypes were selected from our STS PDX platform (n = 45) by AXL immunostaining on archived passages. Models were expanded by unilateral transplantation of tumor tissue into the left flank of 20 NMRI nu/nu mice. Once tumors were established, mice were randomized into an EnaV treatment group, or a group treated with isotype control ADC. Treatment efficacy was assessed by tumor volume evaluation, survival analysis, and histological evaluation of tumors, and associated with AXL expression. EnaV demonstrated significant tumor growth delay, regression, and/or prolonged survival compared to isotype control ADC in 5/8 STS PDX models investigated. Experimental passages of responding models were all found positive for AXL at varying levels, but no linear relationship could be identified between the level of expression and level of response to EnaV. One model was found negative for AXL on experimental passage and did not respond to EnaV. This study provides a preclinical rationale for the evaluation of AXL-targeting ADCs in the treatment of AXL-expressing sarcomas.
Subject(s)
Antineoplastic Agents , Immunoconjugates , Sarcoma , Soft Tissue Neoplasms , Animals , Mice , Antineoplastic Agents/therapeutic use , Disease Models, Animal , Immunoconjugates/therapeutic use , Sarcoma/drug therapy , Sarcoma/pathology , Soft Tissue Neoplasms/drug therapy , Soft Tissue Neoplasms/pathology , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Pediatric MLL-rearranged acute myeloid leukemia (AML) has a generally unfavorable outcome, primarily due to relapse and drug resistance. To overcome these difficulties, new therapeutic agents are urgently needed. Yet, implementing novel drugs for clinical use is a time-consuming, laborious, costly and high-risk process. Therefore, we applied a drug-repositioning strategy by screening drug libraries, comprised of >4000 compounds that are mostly FDA-approved, in a high-throughput format on primary MLL-rearranged AML cells. Here we identified pyrvinium pamoate (pyrvinium) as a novel candidate drug effective against MLL-rearranged AML, eliminating all cell viability at <1000â¯nM. Additional screening of identified drug hits on non-leukemic bone marrow samples, resulted in a decrease in cell viability of â¼50% at 1000â¯nM pyrvinium, suggesting a therapeutic window for targeting leukemic cells specifically. Validation of pyrvinium on an extensive panel of AML cell lines and primary AML samples showed comparable viabilities as the drug screen data, with pyrvinium achieving IC50 values of <80â¯nM in these samples. Remarkably, pyrvinium also induced cell toxicity in primary MLL-AF10+ AML cells, an MLL-rearrangement associated with a poor outcome. While pyrvinium is able to inhibit the Wnt pathway in other diseases, this unlikely explains the efficacy we observed as ß-catenin was not expressed in the AML cells tested. Rather, we show that pyrvinium co-localized with the mitochondrial stain in cells, and hence may act by inhibiting mitochondrial respiration. Overall, this study shows that pyrvinium is highly effective against MLL-rearranged AML in vitro, and therefore represents a novel potential candidate for further studies in MLL-rearranged AML.
ABSTRACT
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.
Subject(s)
Immune Checkpoint Inhibitors/therapeutic use , Immunoconjugates/therapeutic use , Lung Neoplasms/therapy , Melanoma/therapy , Proto-Oncogene Proteins/immunology , Receptor Protein-Tyrosine Kinases/immunology , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Combined Modality Therapy , Drug Resistance, Neoplasm/immunology , Drug Synergism , HEK293 Cells , Humans , Immune Checkpoint Inhibitors/administration & dosage , Immunoconjugates/administration & dosage , Immunotherapy , Lung Neoplasms/pathology , Male , Melanoma/pathology , Mice , Mice, Nude , Mice, Transgenic , Molecular Targeted Therapy/methods , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseSubject(s)
Antimetabolites, Antineoplastic/pharmacology , Deoxycytidine/analogs & derivatives , Gene Rearrangement , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Animals , Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Disease Management , Disease Models, Animal , Drug Evaluation, Preclinical , Genetic Predisposition to Disease , Humans , Infant , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , GemcitabineABSTRACT
MLL-rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL-rearranged ALL cells in vitro. Here, we assessed the in vivo anti-leukemic potential of low-dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL-rearranged ALL. Furthermore, we explored whether prolonged exposure to low-dose decitabine could chemo-sensitize MLL-rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic-based and anti-neoplastic compounds. Our data reveal that decitabine prolonged survival in xenograft mice of MLL-rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out-growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low-dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL-rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic-based or anticancer drugs using high-throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line-dependent manner.
Subject(s)
Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyridones/pharmacology , Pyrimidinones/pharmacology , ras Proteins/genetics , Animals , Cell Line, Tumor , Gene Rearrangement , Heterografts , Humans , MAP Kinase Signaling System , Mice , Phosphorylation/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnostic imaging , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Tumor Burden/drug effectsABSTRACT
INTRODUCTION: MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions. METHODS: For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples. RESULTS: Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples. CONCLUSION: We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.
Subject(s)
Gene Expression Regulation, Leukemic , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Translocation, Genetic , Adult , Cell Line, Tumor , Child, Preschool , Cluster Analysis , DNA Methylation , Female , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Promoter Regions, Genetic , RNA, Small Interfering/genetics , TranscriptomeABSTRACT
SiRNA molecules represent promising therapeutic molecules, e.g. for cancer therapy. However, efficient delivery into tumor cells remains a major obstacle for treatment. Here, we describe a liposomal siRNA carrier system for targeted delivery of siRNA to CD33-positive acute myeloid leukemia cells. The siRNA is directed against the t(8;21) translocation resulting in the AML1/MTG8 fusion protein. The siRNA was encapsulated in free or polyethylene imine (PEI)-complexed form into PEGylated liposomes endowed subsequently with an anti-CD33 single-chain Fv fragment (scFv) for targeted delivery. The resulting siRNA-loaded immunoliposomes (IL) and immunolipoplexes (ILP) showed specific binding and internalization by CD33-expressing myeloid leukemia cell lines (SKNO-1, Kasumi-1). Targeted delivery of AML1/MTG8 siRNA, but not of mismatch control siRNA, reduced AML1/MTG8 mRNA and protein levels and decreased leukemic clonogenicity, a hallmark of leukemic self-renewal. Although this study revealed that further modifications are necessary to increase efficacy of siRNA delivery and silencing, we were able to establish a targeted liposomal siRNA delivery system combining recombinant antibody fragments for targeted delivery with tumor cell-specific siRNA molecules as therapeutic agents.