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Supplementary Data from Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

Key Points Candidate discovery of translational feasibility for noninvasive measurement of miRNA from plasma vesicles as a biomarker for AML patients.Preclinical validation of miR-1246 (candidate among 15 miRNA) as a potential minimally invasive AML biomarker for early monitoring of MRD., Recurrent disease remains the principal cause for treatment failure in acute myeloid leukemia (AML) across age groups. Reliable biomarkers of AML relapse risk and disease burden have been problematic, as symptoms appear late and current monitoring relies on invasive and cost-ineffective serial bone marrow (BM) surveillance. In this report, we discover a set of unique microRNA (miRNA) that circulates in AML-derived vesicles in the peripheral blood ahead of the general dissemination of leukemic blasts and symptomatic BM failure. Next-generation sequencing of extracellular vesicle-contained small RNA in 12 AML patients and 12 controls allowed us to identify a panel of differentially incorporated miRNA. Proof-of-concept studies using a murine model and patient-derived xenografts demonstrate the feasibility of developing miR-1246, as a potential minimally invasive AML biomarker.

Bortezomib (BTZ) was recently evaluated in a randomized phase 3 clinical trial by the Children’s Oncology Group (COG) that compared standard chemotherapy (cytarabine, daunorubicin, and etoposide [ADE]) vs standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia (AML). Although the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefiting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. Total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) were measured in leukemic cells from 483 pediatric patients using reverse phase protein arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared with CD34+ nonmalignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs 67% for low-HSF1-pSer326 treated with ADEB (P = .019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and nonphosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs those with wild-type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients who benefit from BTZ-containing chemotherapy.

Purpose: The addition of the proteasome inhibitor (PI) bortezomib to standard chemotherapy (ADE: cytarabine [Ara-C], daunorubicin, and etoposide) did not improve overall outcome of pediatric AML patients in the Children's Oncology Group AAML1031 phase 3 randomized clinical trial (AAML1031). Bortezomib prevents protein degradation, including RelA via the intracellular NF-kB pathway. In this study, we hypothesized that subgroups of pediatric AML patients benefitting from standard therapy plus bortezomib (ADEB) could be identified based on pre-treatment RelA expression and phosphorylation status.Experimental design: RelA-total and phosphorylation at serine 536 (RelA-pSer536) were measured in 483 patient samples using reverse phase protein array technology.Results: In ADEB-treated patients, low-RelA-pSer536 was favorably prognostic when compared to high-RelA-pSer536 (3-yr overall survival (OS): 81% vs. 68%, p = 0.032; relapse risk (RR): 30% vs. 49%, p = 0.004). Among low-RelA-pSer536 patients, RR significantly decreased with ADEB compared to ADE (RR: 30% vs. 44%, p = 0.035). Correlation between RelA-pSer536 and 295 other assayed proteins identified a strong correlation with HSF1-pSer326, another protein previously identified as modifying ADEB response. The combination of low-RelA-pSer536 and low-HSF1-pSer326 was a significant predictor of ADEB response (3-yr OS: 86% vs. 67%, p = 0.013).Conclusion and clinical relevance: Bortezomib may improve clinical outcome in a subgroup of AML patients identified by low-RelA-pSer536 and low-HSF1-pSer326.

Reverse phase protein arrays (RPPA) can assess protein expression and activation states in large numbers of samples (n>1000) and evidence suggests feasibility in the setting of multi-institution clinical trials. Despite evidence in solid tumors, little is known about protein stability in leukemia. Proteins collected from leukemia cells in blood and bone marrow biopsies must be sufficiently stable for analysis. Using 58 leukemia samples, we initially assessed protein/phospho-protein integrity for the following preanalytical variables: 1) shipping vs local processing, 2) temperature (4°C vs ambient temperature), 3) collection tube type (heparin vs Cell Save (CS) preservation tubes), 4) treatment effect (pre- vs post-chemotherapy) and 5) transit time. Next, we assessed 1515 samples from the Children's Oncology Group Phase 3 AML clinical trial (AAML1031, NCT01371981) for the effects of transit time and tube type. Protein expression from shipped blood samples was stable if processed in ≤72h. While protein expression in pre-chemotherapy samples was stable in both heparin and CS tubes, post-chemotherapy samples were stable in only CS tubes. RPPA protein extremes is a successful quality control measure to identify and exclude poor quality samples. These data demonstrate that a majority of shipped proteins can be accurately assessed using RPPA. SIGNIFICANCE: RPPA can assess protein abundance and activation states in large numbers of samples using small amounts of material, making this method ideal for use in multi-institution clinical trials. However, there is little known about the effect of preanalytical handling variables on protein stability and the integrity of protein concentrations after sample collection and shipping. In this study, we used RPPA to assess preanalytical variables that could potentially affect protein concentrations. We found that the preanalytical variables of shipping, transit time, and temperature had minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables could be successfully managed in a multi-site clinical trial setting.

Introduction: The endoplasmic reticulum (ER) is the major site of protein synthesis and folding in the cell. Three pathways are integrated to maintain ER homeostasis: ER-associated degradation (ERAD), unfolded protein response (UPR), and autophagy. One of the chief elements of ERAD is the highly conserved AAA-ATPase superfamily member valosin-containing protein (VCP, or p97). Various studies have reported an upregulation of VCP in cancer and an association between elevated VCP expression and unfavorable cancer outcome. A promising therapeutic approach relies on targeting the cellular stress response, and systemic functional genomic screens have identified VCP as potential target for inhibition in acute myeloid leukemia (AML). As clinical impact of VCP has not been studied, we wondered whether baseline protein expression levels were predictive of outcome in acute leukemia (AL). Methods: Reverse Phase Protein Array (RPPA) was performed with strictly validated antibodies, including antibodies against VCP, IRE1 and GRP78, to determine the protein expression levels of diagnostic leukemic cells from 500 pediatric AML, 818 adult AML, 268 pediatric T-ALL and 93 adult T-ALL patient samples. Pediatric patients participated on either the COG AAML1031 or AALL1231 clinical trial comparing standard therapy (ADE or AFBM) to standard therapy plus bortezomib (ADE+B or AFBM+B). Adults were treated under a variety of protocols. Pearson correlation analyses was used to identify significant protein-protein correlations. Estimates of survival was calculated using the Kaplan-Meier method. Results: VCP protein was expressed in both AML and T-ALL. Although VCP was slightly more highly expressed in AL vs normal CD34+ cells, the majority of patients had VCP expression within the normal range. In pediatric AML, VCP was more highly expressed in younger patients (< 2 y/o), KMT2A (formerly MLL)-rearrangement (p Conclusion: Using a proteomics approach we identified low-VCP as favorable prognostic indicator. This prognostic association was independent of treatment with a proteasome inhibitor, suggesting that the prognostic effect was potentially separate from the proteasome, and points toward a potential for VCP drug inhibition. Negative correlation with VCP and IRE1 and GRP78 might imply that cells with higher VCP rely on proteasomal degradation, whereas those with low VCP are more dependent on other pathways for protein degradation. Also, adaptation in VCP levels may not be a prominent feature of the stress response to chemotherapy. More information is needed to understand what is driving VCP expression. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: The role of the immune system and the microenvironment in Hodgkin Lymphoma (HL) has been well established. However, little is known about the effects of treatment on the immune cells in the peripheral blood or the frequency of tumor specific T cell responses to non-EBV HL specific antigens in pediatric and adolescent patients with HL. The goal of this project was to evaluate whether patients had increased tumor specific cytotoxic T cell responses and a pro-inflammatory cytokine milieu after treatment compared to at diagnosis. Methods: Peripheral blood samples were collected at diagnosis(pre) and following completion of treatment (post) from newly diagnosed high risk classical HL patients ages ≥2 to 22 years enrolled on a Children's Oncology Group sponsored Phase 3 clinical trial AHOD1331 randomizing to therapy containing the anti-CD30 immunoconjugate Brentuximab(Bv-AVEPC) vs. standard chemotherapy (ABVE-PC) (NCT02166463). Peripheral blood mononuclear cells (PBMC) and plasma were isolated using Ficoll Density gradient and analyzed in 206 patients to date. Pre and post plasma samples from 175 patients were tested for Th1/Th2 cytokines by Luminex assay. Tumor specific T cell responses at pre and post treatment have been tested in 64 of the 175 patients. Briefly, non-adherent T cells were stimulated ex vivo with autologous dendritic cells pulsed with HL specific pepmixes for the non-EBV tumor associated antigens (TAA) MAGEA4, PRAME and Survivin and expanded for a week in the presence of cytokines. TAA specificity was then tested using Interferon-γ ELISPOT assay. Immunophenotyping of the T cell subsets and the exhaustion marker profile was performed by flow cytometry in 49 patients with available PBMCs at both time points. Changes in cytokine levels pre versus post treatment were compared in univariate and multivariate analyses including the following variables: age at diagnosis, gender, stage, B symptoms, bulky disease and EBER status. Results: There was no difference in patient characteristics between the entire study cohort (n=587) and patients analyzed (n=206). There was a significant decrease in peripheral blood helper T cells (CD3+CD4+) and an increase in cytotoxic T cells (CD3+/CD8+) after treatment compared to at diagnosis (n=49 patients; p Conclusions: T cell responses to tumor associated antigens can be detected in patients with HL at diagnosis and after treatment. Increased T cell responses to MAGE4 and PRAME post therapy suggests that recovery post anti-HL treatment can promote tumor antigen specific T cell immunity in vivo. Further, reduced IL-13 and increased Interferon-γ following treatment suggest a favorable milieu for T cell expansion. Correlations between immune responses and clinical outcomes will be performed once outcome data are available. The impact of Bv will be evaluated by comparing the differences of the immune responses by treatment arm. Our results will help identify immune markers of response that can guide future immunotherapies for HL. Disclosures Bollard: Mana Therapeutics: Other: IP.

Background This Phase 2 study tested the tolerability and efficacy of bortezomib combined with reinduction chemotherapy for pediatric patients with relapsed, refractory or secondary acute myeloid leukemia (AML). Correlative studies measured putative AML leukemia initiating cells (AML-LIC) before and after treatment.

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

Purpose Despite the encouraging results of bortezomib (BTZ) in hematologic malignancies to date, resistance to BTZ may be a limiting factor to its efficacy. Hence, parameters that may identify responsiveness to BTZ-containing therapy will be of clinical interest. Recently, we reported that higher ratios of immunoproteasome over constitutive proteasome protein expression in pediatric ALL and AML leukemia cells at diagnosis were an accountable factor for ex vivo sensitivity to proteasome inhibitors (Niewerth et al, Haematologica 2013). Here we explored whether this parameter was associated with response to BTZ in first relapsed and refractory pediatric acute leukemia patients treated in phase II clinical trials of BTZ combined with re-induction chemotherapy for pediatric ALL (COG-AALL07P1) and pediatric AML (COG-AAML07P1). Methods Protein expression levels of constitutive- β5 and β1, and immunoproteasome subunits β5i and β1i were determined by Western blot analysis in 61 acute leukemia patient samples (ALL n=47, AML n=14) obtained before BTZ-containing reinduction therapy. In addition, β5 and β5i proteasome catalytic activities were measured in 14 ALL and 13 AML samples prior to treatment. Lastly, NF-ĸB activity was determined by p65 ELISA in nuclear extracts of PBMCs before and 24h after BTZ treatment. Results In pre-treatment samples, expression ratios of both β5i/β5 and β1i/β1 were significantly higher in ALL cells than in AML cells (P=0.049 and P=0.002, respectively). Ratios of both β5i/β5 and β1i/β1 were significantly higher in patients that reached complete remission (CR; n=39) compared to patients that did not reach CR (n=22) (P=0.009 for β5i/β5, P=0.025 for β1i/β1). Moreover, increased ratios of β5i/β5 catalytic activity were observed in pre-treatment ALL+AML samples that reached CR compared to those that did not reach CR (P=0.078). Proteasome activity ratios correlated significantly with proteasome expression ratios (R=0.55 P=0.005). Notably, NF-ĸB activity was similar in both groups and was suppressed after BTZ treatment, being most pronounced in the pre-B ALL patients that achieved CR (average decrease: 47% p=0.05). Conclusion These results suggest that a higher ratio of immuno/constitutive proteasome in pretreatment ALL and AML cells is an accountable factor for the clinical response to BTZ. These results warrant further investigation to establish a biomarker that can be used for selecting relapsed pediatric acute leukemia patients eligible for BTZ-containing reinduction treatment. This study was sponsored by KiKa (Children Cancer-free-GJLK), Millennium pharmaceuticals (TMH), and NIH-K23-CA113775 (TMH) Citation Format: Denise Niewerth, Gertjan J.L. Kaspers, Gerrit Jansen, Johan van Meerloo, Sonja Zweegman, Gaye Jenkins, James A. Whitlock, Stephen P. Hunger, Xiaomin Lu, Jacqueline Cloos, Terzah M. Horton. Ratios of immunoproteasome over constitutive proteasome expression are an indicator for sensitivity to bortezomib-containing reinduction chemotherapy in pediatric relapsed ALL and AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-169. doi:10.1158/1538-7445.AM2014-LB-169

Background: Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling. Methods/Results: We analyzed 9 viably cryopreserved diagnostic samples collected from infants with MLL-R ALL (n=4 MLL-AF4, n=5 MLL-ENL). Using flow cytometry, we identified 2 phenotypically-defined LIC subpopulations, CD34+CD38+CD19+ and CD34-CD19+ (Aoki et al 2015), within the bulk leukemic blast population (CD45+). Consistent with previous findings, 34+38+19+ LICs were predominant in MLL-AF4 samples, while 34-19+ LICs were predominant in MLL-ENLsamples. Next, we measured surface expression of the adhesion molecule receptors CXCR4, CD49d (VLA-4), CXCR7, and CXCR3 (quantified as mean fluorescence intensity, MFI). Overall, surface expression of CXCR4, CD49d, CXCR7, and CXCR3 was higher in the 34+38+19+ LICs, compared to 34-19+ LICs (e.g., CXCR4 MFI 142 in 34+38+19+ vs. 76 in 34-19+, p=0.02). Next, samples were treated with dose ranges (0-30 µM) of AraC or etoposide (Etop) and cultured for 48 hours in 3 conditions: 1) off stroma, 2) on stroma, or 3) on stroma with the CXCR4 inhibitor AMD3100 (10 μM). Stromal cells were cultured from a healthy BM donor. In vehicle control-treated 34+38+19+ LICs, stromal co-culture led to increased surface expression of CD49d (avg 64% increase, p=0.03) and CXCR7 (avg 67% increase, p=0.02). Conversely, in vehicle control-treated 34-19+ LICs, stromal co-culture did not affect surface expression of the measured adhesion molecule receptors. In all 3 culture conditions, surface expression of CXCR4, CD49d, and CXCR7 was significantly higher in 34+38+19+ LICs compared to 34-19+ LICs (p≤0.01). We then measured apoptosis by flow cytometry and Annexin V binding. In the absence of chemotherapy, stromal co-culture significantly protected 34+38+19+ LICs from spontaneous apoptosis (p Conclusions: Our results suggest that the two immunophenotypically distinct LIC populations in infant MLL-R ALL may rely differently on the BM microenvironment. Higher surface expression of adhesion molecule receptors may explain why 34+38+19+ LICs derive more benefit from stromal co-culture, while lower surface expression of adhesion molecule receptors may explain decreased reliance on stroma by 34-19+ LICs. There may also be differences in chemotherapy resistance between the LIC populations. Despite these differences, CXCR4 inhibition enhanced chemosensitivity in both LIC populations. These results support the development of new therapeutic strategies that target leukemia-stroma interactions to improve outcomes for infants with MLL-R ALL. Disclosures No relevant conflicts of interest to declare.

Background Although AML and ALL are thought to arise from different progenitor cells, (common myeloid vs. common lymphoid) they share many clinical features and pathophysiological characteristics, such as excessive proliferation in association with blocks in differentiation, that raise the question of whether they might share mechanistic commonalities despite separate ontologic origins. This in turn would suggest common therapies to utilize in cases with similar mechanisms of action. Using reverse-phase protein lysate arrays (RPPA), we strove to determine if RPPA could 1) determine protein activation differences between pediatric ALL and AML, and 2) determine if protein expression clusters were affected by sample collection method, an issue critical to assessing protein activation pathways in the setting of cooperative group clinical trials. Methods We generated a custom RPPA with 132 leukemia enriched samples. The array included 59 AML (3 APL, 1 TMD, 1 relapse) and 73 ALL (58 pre-B, 15 T-ALL, 6 relapsed) patients. Median age was 9.1 years (range 0.0 – 22.1 years). Samples were enriched for leukemia cells using ficoll followed by magnetic bead separation, either CD3/CD19 depletion (AML), or B/T-cell isolation (ALL). The RPPA was probed with 194 strictly validated antibodies (139 antibodies assessing total protein expression, 36 phosphoproteins, 6 cleaved forms, and 3 methylation sites). Samples were collected in either heparin tubes (n=101) or CellSave preservations tubes (Veridex) (n=97), with 57 samples having both tube types. Results Clustering demonstrated that AML and ALL have highly different protein expression and activation patterns. Pre-treatment samples collected in Cell Save preservation tubes revealed two protein clusters dividing the patients into two groups based on the levels of 62/194 significantly differentially expressed proteins (p=0.01, FDR = 0.027). An AML-dominant cluster contained 31 AML and 4 ALL among its 35 members. The ALL-dominant cluster had 62 ALL and a lone AML sample among 63 members. Using heparin tubes, four protein clusters were observed which divided patients into three clusters based on the differential expression of 83 proteins (p=0.01, FDR Twenty-seven proteins were different in both the CellSave (CS) and heparin analyses. An additional 34 proteins, mostly epigenetic modifiers and apoptosis pathway proteins (Bcl2, BAD, BAX), were seen in the CS samples. An additional 52 proteins were detected in the heparin group, many related to cell stress pathways. Conclusion Protein expression profiles strongly divided pediatric ALL from AML. Since this classification scheme is dependent on protein expression and functional activation states, it may be possible to further identify patients with different risk characteristics based on protein expression profiles. However, at least 111 of 194 proteins showed similar expression in both AML and ALL suggesting that there may be commonalities in mechanisms shared between leukemias of different lineages. Further analysis of protein functional groups and pathway utilization are in progress to define both differences and commonalities. Figure 1 Figure 1. Disclosures Off Label Use: bortezomib: off label use in pediatric AML as part of a COG clinical trial.

9528 Background: The alkylating agent temozolomide exerts its primary cytotoxic activity through the addition to O6 methyl adducts to guanine residues in DNA, and temozolomide resistance results either from increased expression of methyl-guanine DNA methyltransferase (MGMT), which removes O6 adducts, or from mutations in the mismatch repair system (MRS), which lead to microsatellite instability (MSI). Temozolomide also creates N3 and N7 methyl adducts that are efficiently removed by the base excision repair (BER) system. PARP inhibitors block BER and may potentiate the cytotoxic effects of temozolomide. Methods: The cytotoxicity of temozolomide in combination with the PARP inhibitor ABT-888 was evaluated in vitro in leukemia cell lines and primary leukemia cells using the MTT assay. PARP activity was measured using a commercially available PARP assay. Results: ABT-888 effectively enhanced temozolomide cytotoxicity in cell lines with elevated MGMT (Jurkat and HSB2 T-cell ALL cell lines) or MRS deficiencies (Jurkat and Molt-4 T cell ALL). The temozolomide IC50 decreased from 450 μM to 35 μM at an ABT-888 concentration of 5 μM in the Jurkat T-cell ALL cell line and from 340 μM to 7.5 μM in the HSB2 T-cell line. ABT-888 also enhanced temozolomide cytotoxicity in non-T-cell leukemia subtypes, decreasing the IC50 in the JM1 pre-B ALL cell line from 51 μM to 7.7 μM and decreasing the IC50 from 316 μM to 22 μM in tumor cells obtained from a patient with pre-B ALL. PARP activity was also examined. In contrast, the sensitivity of U937 AML cells to temozolomide showed no effect of the addition of a PARP inhibitor. These results are consistent with the findings that these cells have low or undetectable MGMT and no MSI to suggest MRS mutations. Conclusion: These results suggest that ABT-888 may enhance the cytotoxic activity of temozolomide in leukemia patients whose tumors are resistant to temozolomide because of elevated MGMT expression and mismatch repair defects. No significant financial relationships to disclose.