Papers by David Alagpulinsa
Data from A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
Molecular Cancer Therapeutics, Feb 1, 2016
PARP1/2 are required for single-strand break repair, and their inhibition causes DNA replication ... more PARP1/2 are required for single-strand break repair, and their inhibition causes DNA replication fork collapse and doublestrand break (DSB) formation. These DSBs are primarily repaired via homologous recombination (HR), a high-fidelity repair pathway. Should HR be deficient, DSBs may be repaired via error-prone nonhomologous end-joining mechanisms, or may persist, ultimately resulting in cell death. The combined disruption of PARP and HR activities thus produces synthetic lethality. Multiple myeloma cells are characterized by chromosomal instability and pervasive DNA damage, implicating aberrant DNA repair. Cyclin-dependent kinases (CDK), upstream modulators of HR, are dysregulated in multiple myeloma. Here, we show that a CDK inhibitor, dinaciclib, impairs HR repair and sensitizes multiple myeloma cells to the PARP1/2 inhibitor ABT-888. Dinaciclib abolishes ABT-888induced BRCA1 and RAD51 foci and potentiates DNA damage, indicated by increased gH2AX foci. Dinaciclib treatment reduces expression of HR repair genes, including Rad51, and blocks BRCA1 phosphorylation, a modification required for HR repair, thus inhibiting HR repair of chromosome DSBs. Cotreatment with dinaciclib and ABT-888 in vitro resulted in synthetic lethality of multiple myeloma cells, but not normal CD19 þ B cells, and slowed growth of multiple myeloma xenografts in SCID mice almost two-fold. These findings support combining dinaciclib with PARP inhibitors for multiple myeloma therapy.
medRxiv (Cold Spring Harbor Laboratory), Apr 1, 2024
Hypothesis/Aim: Type 1 diabetes (T1D) is associated with excess coronary artery disease (CAD) ris... more Hypothesis/Aim: Type 1 diabetes (T1D) is associated with excess coronary artery disease (CAD) risk even when known cardiovascular risk factors are accounted for. Genetic perturbation of hematopoiesis that alters leukocyte production is a novel independent modifier of CAD risk. We examined whether there are shared genetic determinants and causal relationships between leukocyte counts, T1D, and CAD. Methods: Genome-wide association studies (GWAS) summary statistics were utilized to perform pairwise linkage disequilibrium score regression (LDSC) and heritability estimation from summary statistics (r-HESS) to respectively estimate the genome-wide and local genetic correlations, and two-sample Mendelian randomization (MR) to estimate the causal relationships between leukocyte counts (335 855 healthy subjects), T1D (18 942 cases, 501 638 controls), and CAD (122 733 cases, 424 528 controls). Results: There was significant genome-wide genetic correlation (rg) between T1D and CAD (rg = 0.088; P = 9.0e-03) and both diseases shared significant genome-wide genetic determinants with eosinophil count (rg(T1D) = 0.093, P = 7.20e-03; rg(CAD) = 0.092, P = 3.68e-06) and lymphocyte count (rg(T1D) = -0.052, P = 2.80e-02; rg(CAD) = 0.1761, P = 1.82e-15). Sixteen independent loci showed stringent Bonferroni significant local genetic correlations between leukocyte counts, T1D and/or CAD. Cis-genetic regulation of the expression levels of genes within loci that are shared between T1D and CAD were associated with both diseases as well as leukocyte counts, including SH2B3, CTSH, MORF4L1, CTRB1, CTRB2, CFDP1, and IFIH1. Genetically predicted lymphocyte, neutrophil, and eosinophil counts were associated with T1D and CAD (lymphocyte odds ratio (OR)T1D = 0.667, P = 2.02e-19; ORCAD =1.085, P = 2.67e-06; neutrophil ORT1D = 0.82, P = 5.63e-05; ORCAD = 1.17, P = 5.02e-14; and eosinophil ORT1D: 1.67, P = 4.45e-25; ORCAD: 1.07; P = 2.02e-03). This study sheds light on shared genetic mechanisms that underlie T1D and CAD, which may contribute to their co-occurrence through regulation of gene expression and leukocyte counts. This study also identifies molecular targets for further investigation for disease prediction and potential drug discovery. What is already known about the subject? • Genetic factors have been shown to contribute to the occurrence of coronary artery disease (CAD) in type 1 diabetes (T1D), but the mechanisms are unknown. • Genetic perturbation of hematopoiesis that alters leukocyte production is associated with CAD risk and clinical observations have documented altered leukocyte counts in T1D. However, it is unknown whether altered leukocyte frequencies contribute to T1D and the co-occurrence of T1D and CAD or these reflect reverse causation. • Do T1D and CAD share genetic determinants with leukocyte counts, and if so, are genetically predicted leukocyte counts associated with risk of T1D or CAD and their cooccurrence? What are the new findings? • T1D and CAD share significant genetic architecture, and both diseases share significant genetic determinants with eosinophil and lymphocyte counts. • Genetically predicted eosinophil, lymphocyte, and neutrophil counts are associated with risk of T1D and CAD. • Genetic regulation of the expression levels of genes in shared loci between T1D and CAD are associated with both diseases and leukocyte counts. • Genetic heritability for T1D is shared with CAD risk and leukocyte counts, and the counts of eosinophils, lymphocytes, and neutrophils are associated with both diseases.
Supplementary Figure 3: Dinaciclib reduces the mRNA transcripts of Rad51, its paralogs and Brca1 in vivo from A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
When tumor volumes had reached the predetermined limit, as indicated in Materials and Methods, th... more When tumor volumes had reached the predetermined limit, as indicated in Materials and Methods, the treatments were repeated for 24 h and tumors excised and immediately preserved in RNAlater. Total RNA was extracted from the tumors and subjected to RT-qPCR as described in the Materials and Methods. Results represent the mean {plus minus} SEM of at least 3 mice from each treatment group. *, **, *** and **** indicate significance of differences from DMSO controls, at p<0.05, 0.01, 0.001 and 0.0001, respectively.
Supplementary Figure 1: Dinaciclib potentiates cytotoxic effects of doxorubicin from A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
MM.1S (A) and H929 (B) cell lines were treated with doxorubicin (DOX; 0‒160 nM) {plus minus}... more MM.1S (A) and H929 (B) cell lines were treated with doxorubicin (DOX; 0‒160 nM) {plus minus} dinaciclib (SCH; 20 nM); viable cell number was assessed by WST-1 after 72h. Mean survival {plus minus} SEM is shown for 3 replicate experiments normalized to the untreated controls, and statistical comparisons were made by 2-tailed paired t-tests against SCH treatment alone (open bar, DOX = 0 nM), with the exception of SCH treatment, which was compared to its control treatment (i.e. filled vs. open bar, at DOX = 0 nM). *, **, *** and **** indicate p<0.05, 0.01, 0.001, and 0.0001, respectively.
Supplementary Figure 2: Dinaciclib induces S-phase accumulation and reduces G2/M-phase accumulation from A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
(A, B) H929 cells were treated as indicated for 24 h and cell-cycle distribution assessed by prop... more (A, B) H929 cells were treated as indicated for 24 h and cell-cycle distribution assessed by propidium iodide (PI) staining as described in Figure 5.
Advances in understanding the molecular basis of clonal hematopoiesis
Trends in Molecular Medicine, 2022
Hematopoietic stem cells (HSCs) are polyfunctional, regenerating all blood cells via hematopoiesi... more Hematopoietic stem cells (HSCs) are polyfunctional, regenerating all blood cells via hematopoiesis throughout life. Clonal hematopoiesis (CH) is said to occur when a substantial proportion of mature blood cells is derived from a single dominant HSC lineage, usually because these HSCs have somatic mutations that confer a fitness and expansion advantage. CH strongly associates with aging and enrichment in some diseases irrespective of age, emerging as an independent causal risk factor for hematologic malignancies, cardiovascular disease, adverse disease outcomes, and all-cause mortality. Defining the molecular mechanisms underlying CH will thus provide a framework to develop interventions for healthy aging and disease treatment. Here, we review the most recent advances in understanding the molecular basis of CH in health and disease.
Dinaciclib, a CDK Inhibitor, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition
Blood, 2014
Multiple myeloma (MM) cells are characterized by genomic instability, implicating aberrant DNA da... more Multiple myeloma (MM) cells are characterized by genomic instability, implicating aberrant DNA damage repair. They exhibit pervasive double strand breaks (DSBs), the most lethal DNA lesions, as indicated by the constitutive abundance of γH2AX foci. DSBs can be repaired through homologous recombination (HR) or nonhomologous end joining (NHEJ). We previously showed elevated HR in MM cells, which were reported to have impaired NHEJ function; they may thus depend on elevated HR for survival. Poly (ADP-ribose) polymerase 1 (PARP1), hyperactivated in MM cells and required for single-strand break (SSB) repair, when inhibited results in accumulation of SSBs that degenerate into lethal DSBs during replication. HR occurs predominantly in the S and G2 cell-cycle phases, and is the preferred route of DSB repair. Should HR be unavailable, as is the case in BRCA-deficient tumor cells, these DSBs persist and/or are repaired by the more error-prone NHEJ, ultimately leading to cell death. Cyclin-dep...
Frontiers in oncology, 2014
We previously reported high expression of RAD51 and increased homologous recombination (HR) rates... more We previously reported high expression of RAD51 and increased homologous recombination (HR) rates in multiple myeloma (MM) cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX) is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did no...
Plasma osteopontin relates to myocardial fibrosis and steatosis and to immune activation among women with HIV
AIDS
American Journal of Transplantation, 2019
Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic β cells... more Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing pancreatic β cells. Logically, replacement of lost β cells with equally functional, insulin-secreting cells would be a cure for T1D. As proof-of-principle, replacement therapy using deceased donor islets has been demonstrated to restore normoglycemia and insulin independence in patients. 1 However, the scarcity of donor
Pharmacology & therapeutics, 2019
Type 1 diabetes (T1D) is a complex multifactorial disease characterized by autoimmune destruction... more Type 1 diabetes (T1D) is a complex multifactorial disease characterized by autoimmune destruction of insulin-producing pancreatic β cells. Our understanding of the pathogenic mechanisms and natural history of T1D has evolved significantly over the past two decades; we can efficiently predict high-risk individuals, early diagnose the disease and stage progression. Fortuitously, novel in vitro differentiation protocols for generating functional β-like cells from human pluripotent stem cells have been developed. These advances provide a definitive roadmap to implement realistic preventive and β-cell replacement therapies in T1D. Immunoprotection and preservation of functional β-cell mass are a sine qua non for the success of these interventions. The chemokine, stromal cell-derived factor-1alpha, known as CXCL12-α, is an attractive therapeutic target molecule in this context. CXCL12-α signaling promotes β-cell development, survival and regeneration and can mediate local immunomodulation...
Cardiac strain is lower among women with HIV in relation to monocyte activation
PLOS ONE
Background Women with HIV (WWH) face heightened risks of heart failure; however, insights on immu... more Background Women with HIV (WWH) face heightened risks of heart failure; however, insights on immune/inflammatory pathways potentially contributing to left ventricular (LV) systolic dysfunction among WWH remain limited. Setting Massachusetts General Hospital, Boston, Massachusetts. Methods Global longitudinal strain (GLS) is a sensitive measure of LV systolic function, with lower cardiac strain predicting incident heart failure and adverse heart failure outcomes. We analyzed relationships between GLS (cardiovascular magnetic resonance imaging) and monocyte activation (flow cytometry) among 20 WWH and 14 women without HIV. Results WWH had lower GLS compared to women without HIV (WWH vs. women without HIV: 19.4±3.0 vs. 23.1±1.9%, P<0.0001). Among the whole group, HIV status was an independent predictor of lower GLS. Among WWH (but not among women without HIV), lower GLS related to a higher density of expression of HLA-DR on the surface of CD14+CD16+ monocytes (ρ = -0.45, P = 0.0475)...
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Papers by David Alagpulinsa