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Little Rock, AR, United States

Alagpulinsa D.A.,ellan Veterans Medical Center | Alagpulinsa D.A.,University of Arkansas for Medical Sciences | Ayyadevara S.,ellan Veterans Medical Center | Ayyadevara S.,University of Arkansas for Medical Sciences | And 2 more authors.
Frontiers in Oncology | Year: 2014

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 not produce significant synergy against normal human CD19+ B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ≥5 foci rose 4-fold, whereas γH2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas γH2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy. © 2014 Alagpulinsa, Ayyadevara and Shmookler-reis.

Alagpulinsa D.A.,ellan Veterans Medical Center | Alagpulinsa D.A.,University of Arkansas for Medical Sciences | Ayyadevara S.,ellan Veterans Medical Center | Ayyadevara S.,University of Arkansas for Medical Sciences | And 3 more authors.
Molecular Cancer Therapeutics | Year: 2016

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-888-induced BRCA1 and RAD51 foci and potentiates DNA damage, indicated by increased γH2AX 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+ 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. © 2015 American Association for Cancer Research.

Alagpulinsa D.A.,ellan Veterans Medical Center | Alagpulinsa D.A.,University of Arkansas for Medical Sciences | Yaccoby S.,University of Arkansas for Medical Sciences | Ayyadevara S.,ellan Veterans Medical Center | And 3 more authors.
Cancer Biology and Therapy | Year: 2015

RAD51-mediated recombinational repair is elevated in multiple myeloma (MM) and predicts poor prognosis. RAD51 has been targeted to selectively sensitize and/or kill tumor cells. Here, we employed a peptide nucleic acid (PNA) to inhibit RAD51 expression in MM cells. We constructed a PNA complementary to a unique segment of the RAD51 gene promoter, spanning the transcription start site, and conjugated it to a nuclear localization signal (PKKKRKV) to enhance cellular uptake and nuclear delivery without transfection reagents. This synthetic construct, (PNArad51_nls), significantly reduced RAD51 transcripts in MM cells, and markedly reduced the number and intensity of de novo and melphalan-induced nuclear RAD51 foci, while increasing the level of melphalan-induced γH2AX foci. Melphalan alone markedly induced the expression of 5 other genes involved in homologous-recombination repair, yet suppression of RAD51 by PNArad51_nls was sufficient to synergize with melphalan, producing significant synthetic lethality of MM cells in vitro. In a SCID-rab mouse model mimicking the MM bone marrow microenvironment, treatment with PNArad51_nls ± melphalan significantly suppressed tumor growth after 2 weeks, whereas melphalan plus control PNArad4µ_nls was ineffectual. This study highlights the importance of RAD51 in myeloma growth and is the first to demonstrate that anti-RAD51 PNA can potentiate conventional MM chemotherapy. © 2015 Taylor & Francis Group, LLC.

Ayyadevara S.,ellan Veterans Medical Center | Ayyadevara S.,University of Arkansas for Medical Sciences | Balasubramaniam M.,University of Arkansas for Medical Sciences | Gao Y.,U.S. Food and Drug Administration | And 4 more authors.
Aging Cell | Year: 2015

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2-6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1-42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72-86% (P < 10-6). In worms expressing Aβ1-42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10-6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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