Targeting integrins in drug-resistant acute myeloid leukaemia.

Acute myeloid leukaemia (AML) continues to have a poor prognosis, warranting new therapeutic strategies. The bone marrow (BM) microenvironment consists of niches that interact with not only normal haematopoietic stem cells (HSC) but also leukaemia cells like AML. There are many adhesion molecules in the BM microenvironment; therein, integrins have been of central interest. AML cells express integrins that bind to ligands in the microenvironment, enabling adhesion of leukaemia cells in the microenvironment, thereby initiating intracellular signalling pathways that are associated with cell migration, cell proliferation, survival, and drug resistance that has been described to mediate cell adhesion-mediated drug resistance (CAM-DR). Identifying and targeting integrins in AML to interrupt interactions with the microenvironment have been pursued as a strategy to overcome CAM-DR. Here, we focus on the BM microenvironment and review the role of integrins in CAM-DR of AML and discuss integrin-targeting strategies. LINKED ARTICLES: This article is part of a themed issue on Cancer Microenvironment and Pharmacological Interventions. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.2/issuetoc.

Altered physical phenotypes of leukemia cells that survive chemotherapy treatment.

The recurrence of cancer following chemotherapy treatment is a major cause of death across solid and hematologic cancers. In B-cell acute lymphoblastic leukemia (B-ALL), relapse after initial chemotherapy treatment leads to poor patient outcomes. Here we test the hypothesis that chemotherapy-treated versus control B-ALL cells can be characterized based on cellular physical phenotypes. To quantify physical phenotypes of chemotherapy-treated leukemia cells, we use cells derived from B-ALL patients that are treated for 7 days with a standard multidrug chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VDL). We conduct physical phenotyping of VDL-treated versus control cells by tracking the sequential deformations of single cells as they flow through a series of micron-scale constrictions in a microfluidic device; we call this method Quantitative Cyclical Deformability Cytometry. Using automated image analysis, we extract time-dependent features of deforming cells including cell size and transit time (TT) with single-cell resolution. Our findings show that VDL-treated B-ALL cells have faster TTs and transit velocity than control cells, indicating that VDL-treated cells are more deformable. We then test how effectively physical phenotypes can predict the presence of VDL-treated cells in mixed populations of VDL-treated and control cells using machine learning approaches. We find that TT measurements across a series of sequential constrictions can enhance the classification accuracy of VDL-treated cells in mixed populations using a variety of classifiers. Our findings suggest the predictive power of cell physical phenotyping as a complementary prognostic tool to detect the presence of cells that survive chemotherapy treatment. Ultimately such complementary physical phenotyping approaches could guide treatment strategies and therapeutic interventions. Insight box Cancer cells that survive chemotherapy treatment are major contributors to patient relapse, but the ability to predict recurrence remains a challenge. Here we investigate the physical properties of leukemia cells that survive treatment with chemotherapy drugs by deforming individual cells through a series of micron-scale constrictions in a microfluidic channel. Our findings reveal that leukemia cells that survive chemotherapy treatment are more deformable than control cells. We further show that machine learning algorithms applied to physical phenotyping data can predict the presence of cells that survive chemotherapy treatment in a mixed population. Such an integrated approach using physical phenotyping and machine learning could be valuable to guide patient treatments.

Artemis inhibition as a therapeutic strategy for acute lymphoblastic leukemia.

As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds (827171, 827032, 826941, and 825226), previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines (LAX56, BLQ5 and LAX7R) and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL.

PI3K Targeting in Non-solid Cancer.

Despite the therapeutic progress, relapse remains a major problem in the treatment of acute lymphoblastic leukemia (ALL). Most leukemia cells that survive chemotherapy are found in the bone marrow (BM), thus resistance to chemotherapy and other treatments may be partially attributed to pro-survival signaling to leukemic cells mediated by leukemia cell-microenvironment interactions. Adhesion of leukemia cells to BM stromal cells may lead to cell adhesion-mediated drug resistance (CAM-DR) mediating intracellular signaling changes that support survival of leukemia cells. In ALL and chronic lymphocytic leukemia (CLL), adhesion-mediated activation of the PI3K/AKT signaling pathway has been shown to be critical in CAM-DR. PI3K targeting inhibitors have been approved for CLL and have been evaluated preclinically in ALL. However, PI3K inhibition has yet to be approved for clinical use in ALL. Here, we review the role of PI3K signaling for normal hematopoietic and leukemia cells and summarize preclinical inhibitors of PI3K in ALL.

In vitro and in vivo effects of AVA4746, a novel competitive antagonist of the ligand binding of VLA-4, in B-cell acute lymphoblastic leukemia.

Treatment of resistant or recurrent acute lymphoblastic leukemia (ALL) remains a challenge. It was previously demonstrated that the adhesion molecule integrin α4, referred to hereafter as α4, mediates the cell adhesion-mediated drug resistance (CAM-DR) of B-cell ALL by binding to vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stroma. In addition, it was previously observed that the blockade of α4 with natalizumab or inhibition using the small molecule antagonist TBC3486 sensitized relapsed ALL cells to chemotherapy. However, α4-targeted therapy is not clinically available for the treatment of leukemia to date. In the present study, the use of a novel non-peptidic small molecule integrin α4 antagonist, AVA4746, as a potential new approach to combat drug-resistant B-ALL was explored. An in vitro co-culture = model of primary B-ALL cells and an in vivo xenograft model of patient-derived B-ALL cells were utilized for evaluation of AVA4746. VLA-4 conformation activation, cell adhesion/de-adhesion, endothelial tube formation, in vivo leukemia cell mobilization and survival assays were performed. AVA4746 exhibited high affinity for binding to B-ALL cells, where it also efficiently blocked ligand-binding to VCAM-1. In addition, AVA4746 caused the functional de-adhesion of primary B-ALL cells from VCAM-1. Inhibition of α4 using AVA4746 also prevented angiogenesis in vitro and when applied in combination with chemotherapy consisting of Vincristine, Dexamethasone and L-asparaginase, it prolonged the survival of ~33% of the mice in an in vivo xenograft model of B-ALL. These data implicate the potential of targeting the α4-VCAM-1 interaction using AVA4746 for the treatment of drug-resistant B-lineage ALL.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.

Preclinical Evaluation of a Novel Dual Targeting PI3Kδ/BRD4 Inhibitor, SF2535, in B-Cell Acute Lymphoblastic Leukemia.

The PI3K/Akt pathway-and in particular PI3Kδ-is known for its role in drug resistant B-cell acute lymphoblastic leukemia (B-ALL) and it is often upregulated in refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing pro-proliferative genes and c-Myc is often overexpressed in cancers. The chromatin regulator BRD4 is required for expression of c-Myc in hematologic malignancies including B-ALL. Previously, combination of BRD4 and PI3K inhibition with SF2523 was shown to successfully decrease Myc expression. However, the underlying mechanism and effect of dual inhibition of PI3Kδ/BRD4 in B-ALL remains unknown. To study this, we utilized SF2535, a novel small molecule dual inhibitor which can specifically target the PI3Kδ isoform and BRD4. We treated primary B-ALL cells with various concentrations of SF2535 and studied its effect on specific pharmacological on-target mechanisms such as apoptosis, cell cycle, cell proliferation, and adhesion molecules expression usingin vitro and in vivo models. SF2535 significantly downregulates both c-Myc mRNA and protein expression through inhibition of BRD4 at the c-Myc promoter site and decreases p-AKT expression through inhibition of the PI3Kδ/AKT pathway. SF2535 induced apoptosis in B-ALL by downregulation of BCL-2 and increased cleavage of caspase-3, caspase-7, and PARP. Moreover, SF2535 induced cell cycle arrest and decreased cell counts in B-ALL. Interestingly, SF2535 decreased the mean fluorescence intensity (MFI) of integrin α4, α5, α6, and ß1 while increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of integrins. Taken together, our data provide a rationale for the clinical evaluation of targeting PI3Kδ/BRD4 in refractory or relapsed B-ALL using SF2535.

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, and they continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of expression of mitochondrial DNA and generation of mitochondrial reactive oxygen species indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, inhibition of OxPhos induced transfer of mitochondria derived from mesenchymal stem cells (MSCs) to AML cells via tunneling nanotubes under direct-contact coculture conditions. Inhibition of OxPhos also induced mitochondrial fission and increased functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, so we used electron microscopy to observe mitochondrial transport to the leading edge of protrusions of AML cells migrating toward MSCs. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased mitochondrial transfer of MSCs to AML cells triggered by OxPhos inhibition. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid Leukemia.

Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.

CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.

Wnt Signaling in the Tumor Microenvironment.

Dysregulated Wnt signaling plays a central role in initiation, progression, and metastasis in many types of human cancers. Cancer development and resistance to conventional cancer therapies are highly associated with the tumor microenvironment (TME), which is composed of numerous stable non-cancer cells, including immune cells, extracellular matrix (ECM), fibroblasts, endothelial cells (ECs), and stromal cells. Recently, increasing evidence suggests that the relationship between Wnt signaling and the TME promotes the proliferation and maintenance of tumor cells, including leukemia. Here, we review the Wnt pathway, the role of Wnt signaling in different components of the TME, and therapeutic strategies for targeting Wnt signaling.

Wnt Signaling in Leukemia and Its Bone Marrow Microenvironment.

Leukemia is an aggressive hematologic neoplastic disease. Therapy-resistant leukemic stem cells (LSCs) may contribute to the relapse of the disease. LSCs are thought to be protected in the leukemia microenvironment, mainly consisting of mesenchymal stem/stromal cells (MSC), endothelial cells, and osteoblasts. Canonical and noncanonical Wnt pathways play a critical role in the maintenance of normal hematopoietic stem cells (HSC) and LSCs. In this review, we summarize recent findings on the role of Wnt signaling in leukemia and its microenvironment and provide information on the currently available strategies for targeting Wnt signaling.

Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging.

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia.

Minimal residual disease (MRD) refers to a chemotherapy/radiotherapy-surviving leukemia cell population that gives rise to relapse of the disease. The detection of MRD is critical for predicting the outcome and for selecting the intensity of further treatment strategies. The development of various new diagnostic platforms, including next-generation sequencing (NGS), has introduced significant advances in the sensitivity of MRD diagnostics. Here, we review current methods to diagnose MRD through phenotypic marker patterns or differential gene patterns through analysis by flow cytometry (FCM), polymerase chain reaction (PCR), real-time quantitative polymerase chain reaction (RQ-PCR), reverse transcription polymerase chain reaction (RT-PCR) or NGS. Future advances in clinical procedures will be molded by practical feasibility and patient needs regarding greater diagnostic sensitivity.

Cadherins, Selectins, and Integrins in CAM-DR in Leukemia.

The interaction between leukemia cells and the bone microenvironment is known to provide drug resistance in leukemia cells. This phenomenon, called cell adhesion-mediated drug resistance (CAM-DR), has been demonstrated in many subsets of leukemia including B- and T-acute lymphoblastic leukemia (B- and T-ALL) and acute myeloid leukemia (AML). Cell adhesion molecules (CAMs) are surface molecules that allow cell-cell or cell-extracellular matrix (ECM) adhesion. CAMs not only recognize ligands for binding but also initiate the intracellular signaling pathways that are associated with cell proliferation, survival, and drug resistance upon binding to their ligands. Cadherins, selectins, and integrins are well-known cell adhesion molecules that allow binding to neighboring cells, ECM proteins, and soluble factors. The expression of cadherin, selectin, and integrin correlates with the increased drug resistance of leukemia cells. This paper will review the role of cadherins, selectins, and integrins in CAM-DR and the results of clinical trials targeting these molecules.

Integrin Antibody Decreases Deformability of Patient-Derived Pre-B Acute Lymphocytic Leukemia Cells as Measured by High-Frequency Acoustic Tweezers.

OBJECTIVES: This article reports a study of cell mechanics in patient-derived (primary) B-cell acute lymphocytic leukemia (ALL) cells treated with antibodies against integrins. Leukemia cell adhesion to stromal cells mediates chemotherapeutic drug resistance, also known as cell adhesion-mediated chemotherapeutic drug resistance. We have previously shown that antibodies against integrin α4 and α6 adhesion molecules can de-adhere ALL cells from stromal cells or counter-receptors. Because drug-resistant cells are more deformable, as evaluated by single-beam acoustic tweezers, we hypothesized that changes in cell mechanics might contribute to the de-adhesive effect of integrin-targeting antibodies. METHODS: In this study, the deformability of primary pre-B ALL cells was evaluated by single-beam acoustic tweezers after treatments with the de-adhering antibody Tysabri or P5G10 against integrin α4 and α6 adhesion molecules. RESULTS: We demonstrated that primary ALL cells treated with P5G10 expressed decreased deformability compared with immunoglobulin G1 -treated control cells (P < .05). Tysabri did not show an effect on deformability (P > .05). CONCLUSIONS: These results suggest that decreased deformability is associated with an integrin α6 blockade. Further assessments of the functional roles of deformability and integrin blockades in B-ALL cell drug resistance and deformability, respectively, are necessary.