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.

The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.

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.

TNB-486 induces potent tumor cell cytotoxicity coupled with low cytokine release in preclinical models of B-NHL.

The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.

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.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies.

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.