ABSTRACT
Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.
Subject(s)
Leukocytes, Mononuclear , Single-Cell Analysis , Humans , Animals , Mice , RNA-Seq , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Algorithms , Gene Expression Profiling/methodsABSTRACT
Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
Subject(s)
Leukemia, Myeloid, Acute , Proto-Oncogene Proteins c-bcl-2 , Humans , Aged , bcl-2-Associated X Protein/genetics , Cell Line, Tumor , Proto-Oncogene Proteins c-bcl-2/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Apoptosis , MutationABSTRACT
Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Indolizines/pharmacology , Isoquinolines/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Myeloid, Acute/drug therapy , Morpholines/pharmacology , Neoplasm Proteins/physiology , Peptide Fragments/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Sulfonamides/pharmacology , Tumor Suppressor Protein p53/physiology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Apoptosis/physiology , Apoptosis Regulatory Proteins/physiology , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , CRISPR-Cas Systems , Cell Line, Tumor , DNA Damage , Genes, p53 , Humans , Indolizines/therapeutic use , Interleukin-2 Receptor alpha Subunit/deficiency , Isoquinolines/therapeutic use , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Morpholines/therapeutic use , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Oxidative Phosphorylation/drug effects , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/administration & dosage , Sulfonamides/therapeutic use , Tumor Suppressor Protein p53/deficiency , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The B-cell lymphoma 2 (BCL-2) inhibitor venetoclax has an emerging role in acute myeloid leukemia (AML), with promising response rates in combination with hypomethylating agents or low-dose cytarabine in older patients. The tolerability and efficacy of venetoclax in combination with intensive chemotherapy in AML is unknown. PATIENTS AND METHODS: Patients with AML who were ≥ 65 years (≥ 60 years if monosomal karyotype) and fit for intensive chemotherapy were allocated to venetoclax dose-escalation cohorts (range, 50-600 mg). Venetoclax was administered orally for 14 days each cycle. During induction, a 7-day prephase/dose ramp-up (days -6 to 0) was followed by an additional 7 days of venetoclax combined with infusional cytarabine 100 mg/m2 on days 1-5 and idarubicin 12 mg/m2 intravenously on days 2-3 (ie, 5 + 2). Consolidation (4 cycles) included 14 days of venetoclax (days -6 to 7) combined with cytarabine (days 1-2) and idarubicin (day 1). Maintenance venetoclax was permitted (7 cycles). The primary objective was to assess the optimal dose schedule of venetoclax with 5 + 2. RESULTS: Fifty-one patients with a median age of 72 years (range, 63-80 years) were included. The maximum tolerated dose was not reached with venetoclax 600 mg/day. The main grade ≥ 3 nonhematologic toxicities during induction were febrile neutropenia (55%) and sepsis (35%). In contrast to induction, platelet recovery was notably delayed during consolidation cycles. The overall response rate (complete remission [CR]/CR with incomplete count recovery) was 72%; it was 97% in de novo AML and was 43% in secondary AML. During the venetoclax prephase, marrow blast reductions (≥ 50%) were noted in NPM1-, IDH2-, and SRSF2-mutant AML. CONCLUSION: Venetoclax combined with 5 + 2 induction chemotherapy was safe and tolerable in fit older patients with AML. Although the optimal postremission therapy remains to be determined, the high remission rate in de novo AML warrants additional investigation (ANZ Clinical Trial Registry No. ACTRN12616000445471).
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Leukemia, Myeloid, Acute/drug therapy , Age Factors , Aged , Aged, 80 and over , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/adverse effects , Cytarabine/administration & dosage , Cytarabine/adverse effects , Dose-Response Relationship, Drug , Female , Humans , Idarubicin/administration & dosage , Idarubicin/adverse effects , Induction Chemotherapy , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Maintenance Chemotherapy , Male , Middle Aged , Nuclear Proteins/genetics , Nucleophosmin , Sulfonamides/administration & dosage , Sulfonamides/adverse effectsABSTRACT
Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.
Subject(s)
Biomimetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Thiophenes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Drug Therapy, Combination , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Peptide Fragments , Proto-Oncogene Proteins , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIX™ cancer vaccine. All patients tested had Treg (CD25(bright) FoxP3(+) CD127(neg)) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1(157-170) epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1(115-132) peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.
Subject(s)
Cancer Vaccines/therapeutic use , Melanoma/immunology , Melanoma/therapy , T-Lymphocytes, Regulatory/immunology , Cells, Cultured , Epitopes/immunology , Flow Cytometry , Humans , Leukocytes, Mononuclear/metabolism , Melanoma/pathologyABSTRACT
Antigenic epitopes recognized by FoxP3(+) regulatory T cells (Treg) are poorly defined, largely due to a lack of assays for determining Treg specificity. We have developed a novel approach for detecting human Treg specific to peptide antigen, utilizing down-regulation of surface CD3 as a read-out of antigen recognition. Culture conditions and re-stimulation time have been optimized, allowing the detection of even very rare Treg, such as those specific to tumor antigens.
Subject(s)
Epitopes, T-Lymphocyte/immunology , Flow Cytometry/methods , T-Lymphocytes, Regulatory/immunology , Antigen Presentation/immunology , CD3 Complex/genetics , CD3 Complex/immunology , Down-Regulation/immunology , Forkhead Transcription Factors/immunology , HumansABSTRACT
Zebrafish are increasingly used to study neurodegenerative conditions such as Parkinson's disease (PD). In rodents, the influence of the genetic background on important experimental parameters in PD research such as susceptibility to toxin exposure or motor behavior is well established. In contrast, little is known about the impact of the genetic background in commonly used zebrafish wild-type strains on these important experimental parameters. We determined the effect of the genetic background in five commonly used zebrafish wild-type strains on crucial, PD-related aspects, in particular the number of ascending dopaminergic neurons, their susceptibility to PD-related neurotoxins, and the expression levels of five genes involved in oxidative stress defense, protein degradation, cell death, and apoptosis. We also investigated whether the susceptibility to morpholino-mediated knockdown of the PD gene DJ-1 may have a varying effect on neuronal cell loss depending on the genetic background. Finally, we determined the influence of the genetic background on spontaneous motor behavior. There was remarkably little variation between the different wild-type strains for most parameters investigated. However, the susceptibility to the neurotoxin 1-methyl-4-phenylpyridinium differed between the five investigated strains and so did their spontaneous motor behavior.