Tatiana Perova

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

Substantial evidence implicates abnormalities of intracellular calcium (Ca2+) dynamics in the pathophysiology of bipolar disorder (BD). However, the precise mechanisms underlying such disturbances are poorly understood. To further elaborate the nature of altered intracellular Ca2+ signalling dynamics that occur in BD, we examined receptor- and store-operated Ca2+ responses in B lymphoblast cell lines (BLCLs), which have been found in earlier studies to 'report' BD-associated disturbances. Basal Ca2+ concentrations ([Ca2+]B), and lysophosphatidic acid (LPA)- and thapsigargin-stimulated Ca2+ responses were determined in BLCLs from 52 BD-I patients and 30 healthy comparison subjects using fura-2, and ratiometric fluorometry. ANOVA revealed a significant effect of diagnosis, but not gender, on [Ca2+]B (F1,63=4.4, p=0.04) and the rate of rise (F1,63=5.2, p=0.03) of LPA-stimulated Ca2+ responses in BLCLs from patients compared with those from healthy subjects. A significant genderxdiagnosis interaction on the LPA-induced rate of rise (F1,63=4.6, p=0.03) was accounted for by a faster rate of rise (97%) in BLCLs from BD-I males compared with healthy males but not in those from female patients compared with healthy females. A genderxdiagnosis interaction in thapsigargin-evoked Ca2+ influx (F1,61=3.8, p=0.05) resulted from a significantly higher peak [Ca2+]influx (24%) in BLCLs from female compared with male patients. The results suggest more rapid LPA-stimulated Ca2+ responses occur in BLCLs from BD-I patients compared with controls, which are probably mediated, in part, by canonical transient receptor potential type 3 (TRPC3)-like channels. Additionally, this study highlights sex-dependent differences that can occur in the pathophysiological disturbances involved in BD.

OBJECTIVES: Disrupted intracellular calcium (Ca(2+) ) homeostasis (ICH) related to mitochondrial and/or endoplasmic reticulum (ER) dysfunction has been implicated in bipolar disorder (BD). The anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2), encoded in a putative BD susceptibility locus, modulates ER-Ca(2+) dynamics. Recently, an intronic single-nucleotide polymorphism (SNP) in the Bcl-2 gene, rs956572, was suggested as a functionally active SNP that influences its messenger RNA (mRNA) and protein level as well as human gray matter volume. We sought to evaluate the impact of this variant on ICH in BD. METHODS: Basal intracellular Ca(2+) concentrations ([Ca(2+) ](B) ) and rs956572 genotypes were determined in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) (n=150), bipolar II disorder (BD-II) (n=65), and major depressive disorder (n=30) patients, and from healthy subjects (n=70). Bcl-2 mRNA and protein levels were determined by quantitative reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Functional interactions of rs956572 with ICH were assessed by thapsigargin- and lysophosphatidic acid (LPA)-stimulated Ca(2+) responses. RESULTS: Although rs956572 variation was not significantly associated with BD, BD-I, or BD-II, BLCL [Ca(2+) ](B) was significantly higher in BD-I G/G patients compared with other genotypes and with healthy subjects. Bcl-2 mRNA and protein levels were lowest in BD-I G/G patients. Compared with A carriers, BD-I patients with G/G variants showed a modest enhancing effect on thapsigargin- and LPA-stimulated Ca(2+) responses. CONCLUSIONS: These findings support the notion that genetic variation in Bcl-2 affecting its expression impacts ICH in BD. Moreover, we show here for the first time that this interactive effect is diagnostically specific to BD-I.

ABSTRACT: Teclistamab, an off-the-shelf B-cell maturation antigen (BCMA) × CD3 bispecific antibody that mediates T-cell activation and subsequent lysis of BCMA-expressing myeloma cells, is approved for the treatment of patients with relapsed/refractory multiple myeloma (R/RMM). As a T-cell redirection therapy, clinical outcomes with teclistamab may be influenced by patient immune fitness and tumor antigen expression. We correlated tumor characteristics and baseline immune profiles with clinical response and disease burden in patients with R/RMM from the pivotal phase 1/2 MajesTEC-1 study, focusing on patients treated with 1.5 mg/kg of teclistamab (N = 165). Peripheral blood samples were collected at screening, and bone marrow samples were collected at screening and cycle 3. Better clinical outcomes to teclistamab correlated with higher baseline total T-cell counts in the periphery. In addition, responders (partial response or better) had a lower proportion of immunosuppressive regulatory T cells (Tregs), T cells expressing coinhibitory receptors (CD38, PD-1, and PD-1/TIM-3), and soluble BCMA and a T-cell profile suggestive of a more cytolytic potential, compared with nonresponders. Neither frequency of baseline bone marrow BCMA expression nor BCMA-receptor density was associated with clinical response to teclistamab. Improved progression-free survival was observed in patients with a lower frequency of T cells expressing exhaustion markers and immunosuppressive Tregs. Overall, response to teclistamab was associated with baseline immune fitness; nonresponders had immune profiles suggestive of immune suppression and T-cell dysfunction. These findings illustrate the importance of the contribution of the immune landscape to T-cell redirection therapy response. This trial was registered at www.ClinicalTrials.gov as #NCT03145181/NCT04557098.

Introduction: Teclistamab is an off-the-shelf, B-cell maturation antigen (BCMA) bispecific IgG4 antibody that redirects CD3+ T cells to mediate T-cell activation and subsequent lysis of BCMA-expressing myeloma cells. The multicohort, open-label, phase 1/2 MajesTEC-1 study is investigating safety/efficacy of teclistamab in patients with relapsed/refractory multiple myeloma (RRMM) who previously received ≥3 lines of therapy. In phase 1, the recommended phase 2 dose (RP2D) of teclistamab was identified as a weekly subcutaneous (SC) dose of 1.5 mg/kg preceded by step-up doses of 0.06 and 0.3 mg/kg. Initial results from patients treated at the RP2D in phase 1/2 (no prior exposure to a BCMA-targeted treatment) demonstrated that teclistamab was well tolerated, with encouraging efficacy. Here, we report translational research data from the pivotal RP2D and active dose cohorts of patients in the MajesTEC-1 study. Methods: Baseline or on-treatment whole blood and bone marrow aspirate samples from pivotal RP2D patients (SC) were analyzed by flow cytometry for BCMA expression or immune populations. Serum samples were analyzed for soluble BCMA (sBCMA) using an electrochemiluminescence ligand binding assay and for cytokines using MSD or Luminex assays. Whole blood from active dose cohorts (intravenous/SC) was analyzed by cytometry by time of flight (CyTOF). Results: Analysis of pivotal RP2D patients demonstrated that the baseline expression of BCMA on bone marrow plasma cells was prevalent and highly variable among patients with RRMM but was not associated with clinical responses to teclistamab. Higher sBCMA levels at baseline were associated with lower response rates and high-risk disease characteristics (higher revised International Staging System stage, high bone marrow plasma cells [>60%], and presence of extramedullary plasmacytoma). Nonresponding patients had lower peripheral CD8 T-cell counts, higher frequency of regulatory T cells (Tregs) and CD38+ Tregs, and higher overall frequencies of T cells that express markers, such as PD-1, TIM-3, and CD38, in both peripheral blood and bone marrow at baseline. Additional analysis at baseline using CyTOF showed the enrichment of memory CD8 T cells expressing PD-1, LAG-3, TIM-3, CD38, and HLA-DR and a higher number of natural killer (NK) cells in nonresponders, as well as an enhancement of a naïve phenotype in T cells in responders. A higher frequency of baseline T cells expressing PD-1, TIM-3, CD38, CD25, as well as PD-1/TIM-3 and PD-1/CD38, was observed in the blood and bone marrow of patients with high bone marrow plasma cells (>60%) and those with high composite tumor score (based on plasmacytosis ≥80%, serum M-spike ≥5 g/dL, and serum free light chain ≥5000 mg/L). Worse progression-free survival (PFS) was associated with higher frequencies of peripheral PD-1+ CD8, CD38+ naïve CD8, CD38+ effector memory CD4, and Tregs at baseline. A higher frequency of baseline CD25+ CD4 and CD38+ CD4 T cells in bone marrow was also correlated with decreased PFS after adjusting for tumor burden. Such a T-cell profile associated with worse clinical outcome likely reflects a dysfunctional and exhausted T-cell phenotype. Consistent with this hypothesis, a lower induction of interferon-gamma and PD-1+ CD8, CD25+ CD4, and CD38+ CD4 T cells was observed with teclistamab treatment in nonresponding patients. Furthermore, teclistamab-mediated induction of peripheral PD-1+ and PD-1+ CD38+ CD8 T cells was lower in patients with high tumor burden. Finally, occurrence of cytokine release syndrome was associated with higher CD3 T-cell frequency and lower baseline sBCMA, TIM-3, and PD-1/TIM-3 expressing CD4 T cells in the periphery. Conclusions: Analysis of baseline correlatives for pivotal RP2D patients suggests an emerging profile for non-responders of unfavorable immune characteristics at baseline, including lower T-cell numbers; higher frequency of T cells at baseline expressing markers such as PD-1, TIM-3, and CD38; higher frequency of Tregs and CD38+ Tregs; and lower proportion of naïve T cells and more NK cells. These data support clinical combinations of teclistamab with agents such as daratumumab or checkpoint inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal