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.
Abstract 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.
Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures.Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset.Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib.We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.
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.
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.
209 Background: Bispecific antibodies that recruit cytotoxic T cells to kill tumor cells are popular due to their targeted mechanism of action. Despite their attractiveness, there are limitations in the clinic due to undesirable toxicities associated with cytokine release. We describe here a platform for generation of a large collection of human anti-CD3 antibodies obtained from custom transgenic rats. Combining these unique anti-CD3 arms with different tumor targeting arms enables creation of bispecific antibodies of varying tumor cell killing capability. These anti-CD3 arms were combined with tumor antigen binding arms, namely, an anti-BCMA arm for multiple myeloma therapy, or an anti-CD22 arm for B-cell acute lymphoblastic leukemia therapy. Methods: Our platform utilizes a discovery approach involving antibody repertoire deep sequencing, high-throughput gene assembly, and recombinant expression, generating a highly diverse panel of antibodies with varying affinities. The CD3 antibodies were tested in in vitro T cell assays using human PBMCs to measure activation and cytokine release. Bispecific antibodies were evaluated for their ability to kill target cell lines upon co-culture with primary human PBMCs. The in vivo efficacy of bispecific antibodies was evaluated in a xenograft mouse model. Results: The in vitro T cell activity of these antibodies as measured by interleukin-2, interferon gamma levels and upregulation of the activation marker CD69 covered a broad spectrum of EC50 values. In co-culture systems with human PBMCs, anti-BCMA or anti-CD22 bispecific antibodies potently killed their respective target expressing cells with varying strengths. Additionally, the cytokine release from T cell activation correlated with the affinity of the anti-CD3 arms. The in vivo efficacy of the bispecific antibodies in a xenograft model with human PBMCs transferred into NSG mice showed striking tumor clearance at a wide range of doses. Conclusions: Our platform is highly suitable for creation of an extensive collection of bispecific antibodies for a variety of disease models by selecting the ideal anti-CD3 arm for each unique tumor antigen binding arm.
