Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

<div><p>Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples <i>in vitro</i> and <i>in vivo</i>. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγ^null mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of <i>BRCA1, CHK1,</i> and <i>RAD51</i> through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of <i>BRCA1, CHK1,</i> and <i>RAD51</i> to enhance cytarabine and daunorubicin sensitivities in AML cells.</p></div

Panobinostat suppresses <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> protein and transcript expression and induces apoptosis in AML cell lines.

<p>THP-1 cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting (<b>Panels A&D</b>). Total RNAs were isolated and mRNA levels were determined by Real-time RT-PCR (<b>Panel B</b>). Apoptotic events were determined by annexin V/PI staining and flow cytometry analysis (<b>Panel C</b>). Protein and mRNA levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> genes were determined by Western blotting (<b>Panel E</b>) and Real-time PCR (<b>Panel F</b>), respectively, in THP-1 cells treated with 10 nM panobinostat for up to 48 h. CTS, OCI-AML3 or U937 AML cells were treated with variable concentrations of panobinostat for 48 h. Whole cell lysates were subjected to Western blotting to measure protein levels for BRCA1, CHK1, and RAD51 in the cells (<b>Panel G</b>). The levels of apoptosis induced by panobinostat were determined by flow cytometry analysis with annexin V/PI staining (<b>Panel H</b>). Transcript levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> genes were determined by Real-time RT-PCR (<b>Panels I–K</b>).</p

Antileukemic activity of panobinostat alone, cytarabine alone, and panobinostat plus cytarabine in a U937 xenograft model.

<p>NOD-SCID-IL2Rγ^null (NSG) mice were injected with luciferase-labeled U937 cells and treated 3 days later with panobinostat (5 mg/kg once daily for 3 weeks), ara-C (6.25 mg/kg once daily for 4 weeks), or combination (panobinostat 5 mg/kg once daily for 3 weeks plus ara-C 6.25 mg/kg once daily for 4 weeks). Serial bioluminescence images of representative mice receiving panobinostat alone (n = 8), ara-C alone (n = 10), or panobinostat plus ara-C (n = 8) (<b>Panel A</b>). When bioluminescence reached 1.5×10⁷ p/s/cm²/sr, day 17 after injection of U937 cells, mice received panobinostat (5 mg/kg once daily×2). Four hours after the second dose mice were sacrificed and Bone Marrows were harvested. Pellets were lysed and subjected to Western Blot (<b>Panel B</b>). Tumor progression monitored by quantitative biophotonic imaging analysis of control and treatment groups (<b>Panel C</b>). A plot of overall survival probability, estimated with the Kaplan–Meier method (<b>Panel D</b>).</p

The roles of BRCA1, CHK1, and RAD51 in cytarabine- or DNR-induced DNA DSBs and apoptosis in THP-1 cells.

<p>THP-1 cells were infected with BRCA1, CHK1, RAD51, or non-target control (NTC) shRNA lentivirus overnight, washed and then treated with 4 µM cytarabine or 25 nM DNR for 48 h. shRNA knockdown of BRCA1, CHK1, or RAD51, and induction of γH2AX by cytarabine or DNR were determined by Western blotting (<b>Panels A–C</b>). The lane headings indicate the treatment conditions ‘Control’ ‘Ara-C’ or ‘DNR’ and the+or – indicate the shRNA-treated cells from which the sample was derived. Apoptotic events in the cells were determined by annexin V/PI staining and flow cytometry analyses (<b>Panel D</b>). THP-1 cells were infected with BRCA1, CHK1, or RAD51 shRNA lentivirus overnight. The cells were washed three times with complete medium and cultured in virus-free complete medium for up to 72 h. The cells were then treated with 50 µM cytarabine or 2 µM DNR for 3 h and the drugs were washed out, and the cells were cultured in drug-free complete medium for up to 8 h. DNA damage was assessed by COMET assay. Representative images at the 8 h time point are shown (<b>Panel E</b>). The median percent DNA in the tail from at least three replicate gels are shown plus or minus the standard error of the mean (Panel F). *indicates p<0.05.</p

A proposed model of molecular mechanisms underlying the cooperative induction of apoptosis by HDACIs and DNA damaging agents in AML cells.

<p>HDACIs suppress the transcript and protein levels of the transcription factor gene <i>E2F1</i>, leading to decreased expression of BRCA1, CHK1, and RAD51. Decreased expression of BRCA1, CHK1, and RAD51 would result in weakened repair of DNA DSBs induced by DNA damaging chemotherapeutic agents (e.g., cytarabine or DNR), thus enhancing apoptosis. At the same time, downregulation of BRCA1 and CHK1 by HDACIs would result in abrogation of cell cycle checkpoint activation (S and/or G2/M) induced by DNA damaging agents, thus forcing cells carrying DNA lesions to progress in the cell cycle and undergo apoptosis.</p

Effects of panobinostat treatment on the transcript levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> genes in diagnostic AML blasts.

<p>Note: Transcript levels for <i>BRCA1</i>, <i>CHK1</i>, and <i>RAD51</i> in panobinostat treated and untreated cells were quantified by real-time RT-PCR and normalized to transcript levels of <i>RPL13A</i>. Results are expressed as means of three independent experiments relative to that of the untreated cells (set as 1).</p

Panobinostat cooperates with cytarabine or DNR in inducing DNA DSBs and apoptosis, and abrogates S and/or G2/M cell cycle checkpoint activation induced by cytarabine or DNR in THP-1 and OCI-AML3 AML cells.

<p>THP-1 or OCI-AML3 cells were treated with cytarabine or DNR, alone or in combination with panobinostat for 48 h. Early and late apoptosis events in the cells were determined by annexin V/PI staining and flow cytometry analyses (<b>Panels A&B</b>). Whole cell lysates were subjected to Western blotting (<b>Panels C&D</b>). Cell cycle distribution was determined by PI staining and flow cytometry analysis (<b>Panels E&F</b>). **indicates p<0.005.</p

Germline <i>ETV6</i> Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

<div><p>Somatic mutations affecting <i>ETV6</i> often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline <i>ETV6</i> p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second <i>ETV6</i> p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type <i>ETV6</i> with retention of the <i>ETV6</i> p. N385fs. Enforced expression of the <i>ETV6</i> mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of <i>ETV6</i> target genes. Our findings highlight a novel role for <i>ETV6</i> in leukemia predisposition.</p></div

Effect of germline ETV6 mutations on transcription.

<p>(a) The effects of germline mutations on ETV6 function were examined using a Dual Luciferase Reporter Assay. Each of the mutants tested exhibited significantly (****P ≤0.0001) impaired transcriptional repression from the <i>PF4</i> and <i>MMP3</i> promoter constructs when contrasted with the WT ETV6 in the co-transfection experiment. The experiment was performed with 6 replicates for each condition and repeated 3 times. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Error of Mean (SEM). (b) The effects of V37M and R181H germline mutations on ETV6 function were examined using a Dual Luciferase Reporter Assay. The experiment was performed with 6 replicates for each condition and repeated twice. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Deviation (SD). (c) Quantitative PCR of ETV6 transcriptional targets <i>EGR1</i> and <i>TRAF1</i> showed reduced transcriptional abundance in the mutants when contrasted with the WT. The effect was most pronounced in the frameshift mutant. The experiment was performed in triplicate for each condition and repeated three times. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Error of Mean (SEM).</p

Identification of germline mutations in <i>ETV6</i> in 2 unrelated kindreds.

<p>(a) In Kindred 1, targeted sequencing identified a germline <i>ETV6</i> L349P mutation. Sequencing was performed on 9 individuals including the proband (arrow) affected with thrombocytopenia and/or ALL and 7 unaffected individuals as noted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005262#pgen.1005262.t001" target="_blank">Table 1</a>. (b) In Kindred 2, clinical whole exome sequencing was performed on the proband (arrow) with ALL, MDS and AML, the mother with thrombocytopenia as well as the unaffected father. An <i>ETV6</i> N385fs mutation was identified. In both kindreds, the <i>ETV6</i> mutations segregated with disease.</p