Grace Shimin Koh

Identification of prognostic protein biomarkers in childhood acute lymphoblastic leukemia (ALL).

Early response to 7 days of prednisolone (PRED) treatment is one of the important prognostic factors in predicting eventual outcome in childhood acute lymphoblastic leukemia (ALL). Using proteomic tools and clinically important leukemia cell lines (REH, 697, Sup-B15, RS4; 11), we have identified potential prognostic protein biomarkers as well as discovered promising regulators of PRED-induced apoptosis. After treatment with PRED, the four cell lines can be separated into resistant (REH) and sensitive (697, Sup-B15, RS4;11). Two dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS identified 77 and 17 significantly differentially expressed protein spots (p<0.05) in PRED-sensitive and PRED-resistant cell lines respectively. Several of these were validated by Western blot including proliferating cell nuclear antigen (PCNA), cofilin 1, voltage-dependent anion-channel protein 1 (VDAC1) and proteasome activator subunit 2 (PA28β). PCNA is a promising protein because of its important roles both in cell cycle regulation and survival control. We subsequently validated PCNA in 43 paired bone marrow samples from children with newly diagnosed ALL (Day 0) and 7 days after PRED treatment (Day 8). ROC curve analysis confirmed that PCNA was highly predictive of PRED response in patients (AUC=0.81, p=0.007) and most interestingly, independent of the molecular subtype, providing a promising universal prognostic marker.

BIM is a prognostic biomarker for early prednisolone response in pediatric acute lymphoblastic leukemia

OBJECTIVE Glucocorticoids such as prednisolone (PRED) are widely used in the treatment of pediatric acute lymphoblastic leukemia. In PRED-induced apoptosis, Bcl-2 family members play important regulatory roles. However, the exact members involved remain unknown. In this study, the roles of Bcl-2 family members in PRED-induced apoptosis and their prognostic value to day 8 PRED response are evaluated. MATERIALS AND METHODS Four clinically important acute lymphoblastic leukemia cell lines, three PRED-sensitive (697, Sup-B15, and RS4;11) and one PRED-resistant (REH) were studied. Thirty paired patient bone marrow samples were obtained at diagnosis (day 0) and after 7 days (day 8) of PRED monotherapy. Twenty-five patients had PRED good response and five PRED poor response. Differential expressions of Bcl-2 members were observed in those samples and BIM was further investigated using gene silencing technology in representative cell line Sup-B15. RESULTS The proapoptotic BH3-only Bcl-2 family member BIM was upregulated only in PRED-sensitive cells. Receiver operating characteristic curve analysis showed that BIM expression was highly predictive of PRED response (area under the curve = 0.81; p = 0.032) in paired patient bone marrow samples and is, most excitingly, independent of molecular subtype. Patients whose BIM protein expression levels fail to upregulate at day 8 compared to day 0 (D8/D0 ratio <0.93) have significantly poorer event-free survival (60%) than those patients whose BIM protein expression levels did upregulate (92%). By silencing BIM in PRED-sensitive cells, PRED-induced apoptosis was inhibited. CONCLUSIONS Upregulation of BIM by PRED in acute lymphoblastic leukemia cells regardless of molecular subtype is significantly prognostic of outcomes, confirming BIMs essential regulatory role in the PRED-induced apoptosis.

Deregulated MIR335 that targets MAPK1 is implicated in poor outcome of paediatric acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Although 90% of patients are now long‐term survivors, the remaining 10% have poor outcome predominantly due to drug resistance. In this study, we carried out genome‐wide microRNA (miRNA) microarray analysis on diagnostic bone marrow samples to determine miRNA expression profiles associated with poor outcome in ALL. A reduced expression of MIR335 was identified as the most significant miRNA abnormality associated with poor outcome. It is well known that glucocorticoid (GC) resistance is one of the major reasons contributing to poor outcome. We show that exogenous expression of MIR335 in ALL cells increases sensitization to prednisolone‐mediated apoptosis. Moreover, we demonstrate that MAPK1 is a novel target of MIR335, and that MEK/ERK inhibitor treatment enhanced prednisolone‐induced cell death through the activation of BIM (BCL2L11). These results provide a possible underlying molecular mechanism to explain the association between reduced MIR335 with poor clinical outcome, and suggest that approaches to re‐introduce MIR335 expression or override MAPK1 activity may offer promising therapeutic strategies in the treatment of ALL.

Abstract 822: Drug resistance towards vincristine in acute lymphoblastic leukemia is mediated by the PI3K-Akt pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Contemporary acute lymphoblastic leukemia (ALL) therapy involves the use of powerful anti-mitotic agents such as vincristine (VCR). VCR targets the microtubule cytoskeleton and disrupts important cellular processes, leading to cell death. Nonetheless, around 20% of patients relapse and become resistant to most drugs. At present, mechanisms of resistance toward VCR remain elusive despite its extensive clinical use, warranting a more in-depth study with respect to ALL. Five VCR-resistant (VCR-R) subclones were established by gradually exposing five ALL VCR-sensitive (VCR-S) cell lines (Jurkat, REH, SEM, RS4;11, 697) to incremental doses of VCR. Comparing resistant to sensitive cells, the IC50 at 48h increased 3-6 logs. Both VCR-S and VCR-R cell lines were subjected to cell cycle analysis by flow cytometry using propidium iodide staining. After a 48h exposure to VCR, cell death for VCR-S cells was the result of a typical G2/M phase arrest. In contrast, a G2/M phase arrest did not precede the VCR-induced cell death of VCR-R cells. VCR-R subclones were also observed to be more resistant to vinblastine, an alternative tubulin binding agent. In addition, there was increased resistance towards other chemotherapeutic drugs such as prednisolone, dexamethasone, daunorubicin and doxorubicin, indicating that resistance to VCR may contribute to cross-resistance after a prolonged incubation. The PI3K/Akt/mTOR signaling pathway is known to play important roles in cancer cell proliferation and survival. The combination of inhibitors with other chemotherapeutic agents also has the potential to enhance leukemic cell death. To further investigate if the PI3K/Akt/mTOR signaling pathway is involved in VCR resistance, we screened a panel of twelve kinase inhibitors (Merck) which selectively targets this pathway. Interestingly, PDK1/Akt/Flt Dual Pathway Inhibitor KP372-1, was highly potent toward all cell lines with IC50 less than 100nM. Our results also showed that the administration of PI3Kα Inhibitor VIII, Akt Inhibitor IV and PI-103, as well as pan-selective PI3K inhibitors Wortmannin and LY294002 in combination with VCR was able to promote increased levels of cell death. Cell cycle analysis also revealed that the combination treatment elicits different methods of cell death between VCR-S and VCR-R cells. This is especially evident upon the co-treatment with pan-selective inhibitors. Optimising the sensitivity of ALL cells and/or reversing resistance to VCR promise to maximise the therapeutic value of this important chemotherapy. This study provides evidence that VCR resistance potentially contributes to cross-resistance. Our data also shows that the PI3K-Akt pathway may be involved in VCR resistance in ALL, providing a valid rationale for further studies and the future application of inhibitors in clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 822. doi:1538-7445.AM2012-822

Pediatric Acute Lymphoblastic Leukemia: Role of BIM Protein in Prednisolone-Induced Apoptosis

Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer characterized by abnormal accumulation of immature blasts in the bone marrow. Glucocorticoids such as prednisolone (PRED) have been widely used in the treatment of pediatric ALL and the resistance to PRED is associated with unfavorable outcome in patients. We have identified BIM to be an important regulator of PRED-induced apoptosis, and its expression level may have prognostic value. By understanding the molecular basis of PRED-induced apoptosis, we hope that improved treatment strategies can be defined.

Abstract 3227: GX15-070 induces cell death in acute lymphoblastic leukemia (ALL) cells by regulating cellular cholesterol metabolism

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: With the improvement of treatment outcome in pediatric acute lymphoblastic leukemia (ALL), about 80% of the patients can be cured. However, around 10% of patients have poor outcome due to drug resistance which calls for novel drugs such as BH3-mimetics. We have previously demonstrated that GX15-070 (GX), a BH3-mimetic, could effectively induce cell death. In this study, we investigate the mechanisms of GX induced cell death in ALL cells. Method: Seven ALL cell lines were used in this study. Cell viability was determined by MTS assay (Promega). Western blot was used to detect protein expression level. Cholesterol (CHO) level was measured by Amplex® Red Cholesterol Assay Kit (Promega). RT-PCR was done by Roche Light Cycler using SYBR Green. Results: There was a dose-dependent cell death induced in all 7 ALL cell lines after treatment with GX. Interestingly, genes that are involved in the CHO synthesis pathway such as LDLR, HMGCR, HMGSC1 and SQLE were upregulated after GX treatment. Furthermore, GX mediated reduction in cellular CHO levels was observed. To verify that CHO depletion could induce cell death in ALL cells, Lovastatin was applied. Our results showed that Lovastatin not only induced a dose-dependent cell death in all 7 ALL cell lines but also upregulated the genes mentioned above. Co-treatment of 0.1µM GX and 25µM Lovastatin could enhance the cell death by further decreasing CHO level. In addition, GX-induced cell death could also be enhanced by 10mM 2-Deoxy-D-glucose (2-DG) co-treatment. 2-DG is an inhibitor of glycolysis which provides metabolites for CHO metabolism. Next, to mimic a culture condition whereby CHO is depleted, we incubated the cells in HBSS without Fetal bovine serum (FBS) for 24h. This CHO depleted condition decreased the cellular CHO level to 46% compared to the cells that were cultured in 10% FBS and RPMI medium. After incubation in HBSS for 24h, 0.1µM GX could eliminate more cells (80%) compared to the same dose of GX in serum supplemented RPMI medium (20%). Both HBSS incubation and GX treatment caused a decrease in anti-apoptotic protein Mcl-1 expression levels, while pro-apoptotic protein BIM and cleaved-PARP protein levels were upregulated. The two treatments did not alter the protein levels of other Bcl-2 family members. These results indicate that Mcl-1 expression is tightly controlled by cellular metabolism and CHO depletion can result in cell death. The mechanism of GX induced cell death may entail the suppression of Mcl-1 expression, hence sensitizing ALL cells to apoptosis. Conclusion: GX can regulate CHO metabolism to induce cell death in ALL cells which are normally dependent on aerobic glycolysis. This highlights the possibility of manipulating CHO metabolism to provide a specific way to treat leukemic cells. The administration of GX in the clinic, alone or in combination, may improve the treatment outcome of leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3227. doi:1538-7445.AM2012-3227

Abstract 2563: Combination of triptolide and prednisolone to induce apoptosis in acute lymphoblastic leukemia cells

Background: Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children. Although considered highly curable, about 20% of children with ALL relapse and majority of them will die of the resistant disease. Sensitivity to the glucocorticoid, prednisolone (PRED), is critical for cure and the development of combination therapy with PRED may improve treatment outcome. Triptolide, a diterpene triepoxide extracted from the Chinese plant Tripterygium wilfordii has been reported to be immunosuppressive, anti-inflammatory, and anti-proliferative in a broad spectrum of diseases. In this study, we investigate the effect of triptolide to induce apoptosis in ALL cells in combination with prednisolone. Methods: Three clinical important leukemia cells lines 697, Sup-B15 and RS4;11 that represent TCF3-PBX1, BCR-ABL1 and MLL-AF4 respectively were used. They were exposed to triptolide as well as in combination with PRED. Cell viability was determined by MTT assay (Promega). Cell Death Detection ELISA (Roche) was used for measuring apoptosis levels. Western blot was used to detect protein expression levels. Caspase-3 and -9 activities were measured using Caspase-Glo Assay kits (Promega). Cell cycle analysis was performed by Propidium Iodide (PI) staining using flow cytometry. Results: Triptolide can induce cell death at ED50 of 15nM for 697, 13nM for Sup-B15, and 13nM for RS4;11 at 24h time point. The ED50 further decreased to 3nM for 697, 4nM for Sup-B15 and 5nM for RS4;11 at 48h time point. Apoptosis induction and cell cycle arrest was observed upon triptolide treatment in these three cell lines. The levels of caspase-3 and caspase-9 were significantly increased with triptolide treatment, suggesting that triptolide induced apoptosis was caspase-dependent. Although PRED treatment as a single agent can also induce apoptosis, cell cycle arrest and caspase activation in these three cell lines, the co-treatment of PRED and triptolide significantly enhanced these effects compared to single treatment (p Conclusion: Triptolide can induce apoptosis in ALL cell lines. Combined therapy with PRED and triptolide is a novel promising therapy for this hematological malignancy that warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2011-2563

Abstract 1033: Prednisolone induces BIM expression in pediatric acute lymphoblastic leukemia and synergizes with BH3-mimetics GX15-070 and ABT-737

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Chemotherapeutic regimens for pediatric acute lymphoblastic leukemia (ALL) treatment entail the use of glucocorticoids such as prednisolone (PRED). Around 5% of patients fail to attain remission due to drug resistance. We have previously shown that the pro-apoptotic BH3-only BCL-2 family member BIM was up-regulated in only in PRED-sensitive cells. After the specific silencing of BIM by siRNA, inhibition of apoptosis was observed and validated by levels of cleaved PARP and caspase activity, providing a clear indication of BIMs significance in PRED induced apoptosis. In this study, protein expression levels of BIM were further evaluated using patient bone marrow. We also examined the ability of two BH3 mimetics, GX15-070 (obatoclax) and ABT-737, to induce apoptosis in ALL cell lines and the effect of drug combinations. Patient bone marrow samples were obtained at diagnosis (Day 0) and after 7 days (Day 8) of PRED monotherapy. Response to PRED was defined as Good (PGR, peripheral blast count < 1000 /µl), and Poor (PPR, peripheral blast count ≥1000 /µl). Twenty-five PGR and five PPR patients were included in this study. Using western immunoblotting, the protein expression of BIM was reproducibly observed in patient samples. The value of ODBim/ODActin at Day 8 was significantly higher than that on Day 0 (p-Value=0.0006) for PGR patients. In contrast, this value did not differ greatly in PPR patients. At Day 0, BIM expression levels of the entire patient cohort was comparable, but levels were significantly different at Day 8 (p-Value=0.01). These results indicate that the upregulation of BIM was induced by PRED in ALL patients and further confirms the essential role of BIM in this apoptotic pathway. Treatment with GX15-070 and ABT-737 as single agents yielded an increase in cell death in a dose dependent manner both in PRED-sensitive and -resistant cell lines. Apoptosis induced by both GX15-070 and ABT-737 could be inhibited by caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK). Simultaneous exposure of ALL cells to PRED and GX15-070 or ABT-737 resulted in synergistic levels of cell death in SUP-B15 and RS4;11 cell lines with combination index (CI) values that are significantly less than one (as calculated by CalcuSyn software version 2.1). This highlights the possibility of enhancing cell death in ALL cells even with a reduction in drug dosage. In conclusion, BH3-only BCL-2 family member BIM, is vital for PRED-induced apoptosis in ALL. BH3 mimetics GX15-070 and ABT-737 can induce apoptosis in ALL cells regardless of PRED response. The synergism between PRED and BH3-mimetics is likely to minimize severe side effects, and more importantly, provide alternative therapies especially for patients that are resistant to conventional chemotherapeutic drugs in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1033.