ABSTRACT
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Subject(s)
Histone Acetyltransferases/metabolism , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Structure, TertiaryABSTRACT
Background: The kinetics of circulating tumor DNA (ctDNA) release following commencement of radiotherapy or chemoradiotherapy may reflect early tumour cell killing. We hypothesised that an increase in ctDNA may be observed after the first fraction of radiotherapy and that this could have clinical significance. Materials and methods: ctDNA analysis was performed as part of a prospective, observational clinical biomarker study of non-small cell lung cancer (NSCLC) patients, treated with curative-intent radiotherapy or chemoradiotherapy. Blood was collected at predefined intervals before, during (including 24 h after fraction 1 of radiotherapy) and after radiotherapy/chemoradiotherapy. Mutation-specific droplet digital PCR assays used to track ctDNA levels during and after treatment. Results: Sequential ctDNA results are available for 14 patients with known tumor-based mutations, including in EGFR, KRAS and TP53, with a median follow-up of 723 days (range 152 to 1110). Treatments delivered were fractionated radiotherapy/chemoradiotherapy, in 2-2.75 Gy fractions (n = 12), or stereotactic ablative body radiotherapy (SABR, n = 2). An increase in ctDNA was observed after fraction 1 in 3/12 patients treated with fractionated radiotherapy with a complete set of results, including in 2 cases where ctDNA was initially undetectable. Neither SABR patient had detectable ctDNA immediately before or after radiotherapy, but one of these later relapsed systemically with a high detected ctDNA concentration. Conclusions: A rapid increase in ctDNA levels was observed after one fraction of fractionated radiotherapy in three cases. Further molecular characterization will be required to understand if a "spike" in ctDNA levels could represent rapid initial tumor cell destruction and could have clinical value as a surrogate for early treatment response and/or as a means of enriching ctDNA for mutational profiling.
ABSTRACT
There is limited knowledge on the benefit of the α-subunit-specific PI3K inhibitor alpelisib in later lines of therapy for advanced estrogen receptor-positive (ER+) HER2- and triple-negative breast cancer (TNBC). We conducted a phase II multicohort study of alpelisib monotherapy in patients with advanced PI3K pathway mutant ER+HER2- and TNBC. In the intention-to-treat ER+ cohort, the overall response rate was 30% and the clinical benefit rate was 36%. A decline in PI3K pathway mutant circulating tumor DNA (ctDNA) levels from baseline to week 8 while on therapy was significantly associated with a partial response, clinical benefit, and improved progression-free-survival [HR 0.24; 95% confidence interval (CI), 0.083-0.67, P = 0.0065]. Detection of ESR1 mutations at baseline in plasma was also associated with clinical benefit and improved progression-free survival (HR 0.22; 95% CI, 0.078-0.60, P = 0.003). SIGNIFICANCE: Alpelisib monotherapy displayed efficacy in heavily pretreated ER+ breast cancer with PIK3CA mutations. PIK3CA mutation dynamics in plasma during treatment and ESR1 mutations detected in plasma at baseline were candidate biomarkers predictive of benefit from alpelisib, highlighting the utility of ctDNA assays in this setting. This article is highlighted in the In This Issue feature, p. 2007.
Subject(s)
Breast Neoplasms , Triple Negative Breast Neoplasms , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Class I Phosphatidylinositol 3-Kinases/genetics , Female , Humans , Mutation , Phosphatidylinositol 3-Kinases/genetics , Receptor, ErbB-2/genetics , Thiazoles , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/geneticsABSTRACT
There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
Subject(s)
F-Box Proteins , Leukemia, Myeloid, Acute , Alcohol Oxidoreductases , DNA-Binding Proteins , F-Box Proteins/genetics , HLA Antigens/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/therapy , Lymphocyte Activation , Protein-Arginine N-Methyltransferases/metabolism , Recurrence , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
People with human immunodeficiency virus (HIV) have higher rates of certain comorbidities, particularly cardiovascular disease and cancer, than people without HIV1-5. In view of observations that somatic mutations associated with age-related clonal hematopoiesis (CH) are linked to similar comorbidities in the general population6-10, we hypothesized that CH may be more prevalent in people with HIV. To address this issue, we established a prospective cohort study, the ARCHIVE study (NCT04641013), in which 220 HIV-positive and 226 HIV-negative participants aged 55 years or older were recruited in Australia. Demographic characteristics, clinical data and peripheral blood were collected to assess the presence of CH mutations and to identify potential risk factors for and clinical sequelae of CH. In total, 135 CH mutations were identified in 100 (22.4%) of 446 participants. CH was more prevalent in HIV-positive participants than in HIV-negative participants (28.2% versus 16.8%, P = 0.004), overall and across all age groups; the adjusted odds ratio for having CH in those with HIV was 2.16 (95% confidence interval 1.34-3.48, P = 0.002). The most common genes mutated overall were DNMT3A (47.4%), TET2 (20.0%) and ASXL1 (13.3%). CH and HIV infection were independently associated with increases in blood parameters and biomarkers associated with inflammation. These data suggest a selective advantage for the emergence of CH in the context of chronic infection and inflammation related to HIV infection.
Subject(s)
Cardiovascular Diseases/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA-Binding Proteins/genetics , HIV Infections/genetics , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Aged , Aging/genetics , Aging/pathology , Cardiovascular Diseases/complications , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/virology , Clonal Hematopoiesis/genetics , DNA Methyltransferase 3A , Dioxygenases , Female , HIV/pathogenicity , HIV Infections/complications , HIV Infections/epidemiology , HIV Infections/virology , Humans , Inflammation/genetics , Inflammation/pathology , Inflammation/virology , Male , Middle Aged , Mutation/genetics , Neoplasms/complications , Neoplasms/epidemiology , Neoplasms/genetics , Neoplasms/virologyABSTRACT
The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Histone Acetyltransferases/antagonists & inhibitors , Immunologic Factors/pharmacology , Molecular Targeted Therapy , Transcription Factors/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Immune System Diseases/drug therapy , Immunologic Factors/chemistry , Immunologic Factors/therapeutic use , Inflammation/drug therapy , Neoplasms/drug therapy , Protein Domains/drug effects , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Trans-Activators/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Bone Marrow/pathology , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Epigenesis, Genetic/drug effects , Female , HEK293 Cells , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Sequence Analysis, RNA , Single-Cell Analysis , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic/drug effects , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0-8) were treated with daily tamoxifen (20 mg) and venetoclax (200-800 mg). Apart from uncomplicated "on-target" lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected ESR1 mutations. SIGNIFICANCE: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors.See related commentary by Drago et al., p. 323.This article is highlighted in the In This Issue feature, p. 305.