DHFR and MSH3 co-amplification in childhood acute lymphoblastic leukaemia, in vitro and in vivo

Abstract Abstract 1887 Further improvements in outcome for childhood acute lymphoblastic leukaemia (ALL) will require a better understanding of the underlying biology of this disease and the fundamental mechanisms of drug resistance. The discoveries that a few populations can initiate leukemia in mouse models and that new populations of leukaemia initiating cells (LIC) can be detected following an initial round of transplantation in these models raises important questions about the biology of the leukaemias. If several cell populations have LIC properties, what are the relationships of these populations to each other and which populations are most important to target with therapy? It will also be important to determine whether there is any correlation in the biological properties of LIC identified in the model systems with the response of the patients to therapy. Assessment of minimal residual disease (MRD) levels provides a sensitive measurement of early treatment response and permits detection of the in vivo selected drug resistant population. CD58 (leucocyte function-associated antigen 3; LFA-3) is a useful marker in MRD tracking of B cell precursor (BCP) ALL. CD58 is over expressed in these cases permitting discrimination of leukaemia blasts from normal B cells. In this study we investigated whether CD58 is expressed on LIC populations in childhood ALL. Expression of CD58 and CD34 was assessed in a cohort of 12 diagnostic samples with mixed prognoses and compared to levels detected in 11 normal bone marrow (NBM) samples. Levels of CD58 were significantly higher in the ALL cases (57.4±37.7%) than on NBM cells (21.1±12.2%; p=0.007). Likewise, the CD34+/CD58+ population was larger in ALL cases than in normal cells (22.2±34.7% and 0.25±0.25%, respectively; p=0.05). Cells from eight of the 12 patients, were sorted on the basis of expression or lack of expression of these markers and the functional ability of the sorted subpopulations was assessed in vitro and in vivo. On sorting, the majority of cells were CD34−/CD58− (43.7±39.2%), 20.7±30.7% were CD34−/CD58+, 19±14.3% were CD34+/CD58+ and the CD34+/CD58− population accounted for 16.6±35.3%. Unsorted cells and all 4 sorted populations were set up in long-term culture to assess proliferative capability and the in vivo propagating potential was assessed in NSG mice. All 4 sorted subpopulations proliferated over the 6 week period but the highest levels of expansion were observed in the cultures of CD34+/CD58+ (6–420 fold) and CD34+/CD58− (3–24 fold) cells. Cytogenetic analyses confirmed that leukaemia cells were maintained in the culture system. Results from the in vivo analyses on 5 cases to date indicate that all 4 subpopulations contain LIC. In these cases, higher levels of engraftment were observed with CD34+/CD58+ (up to 20%) and with CD34−/CD58− subpopulations (6.1-98%). Serial transplantation studies will determine whether there are differences in the repopulating and self-renewal abilities of these LIC. These findings suggest that using CD58 alone or in combination with CD34 would be insufficient to track disease progression in ALL. Incorporating additional markers that are commonly used in MRD panels will provide valuable information on LIC populations and facilitate development of improved disease monitoring. Disclosures: No relevant conflicts of interest to declare.

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Combining BCL-2 Inhibitors with Parthenolide to Treat Childhood Acute Lymphoblastic Leukaemia

Blood ◽

10.1182/blood.v126.23.2531.2531 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2531-2531

Author(s):

Paraskevi Diamanti ◽

William J Barendt ◽

Benjamin C Ede ◽

Charlotte V. Cox ◽

Allison Blair

Keyword(s):

Clinical Trials ◽

Drug Treatment ◽

Acute Lymphoblastic Leukaemia ◽

Lymphoblastic Leukaemia ◽

Conflicts Of Interest ◽

Minimal Risk ◽

Childhood All ◽

Childhood Acute Lymphoblastic Leukaemia

Abstract Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 90% in recent years. Despite these successes, around 15% of patients die of relapse. It is possible that ALL may be maintained by subpopulations of cells, known as leukaemia stem cells (LSC), that are resistant to therapy and subsequent relapses may arise from these cells. Parthenolide (PTL), a naturally occurring sesquiterpene lactone, is an NF-κB inhibitor that kills leukaemia cells by apoptosis and/or increase of reactive oxygen species. PTL has been shown to be remarkably effective against several LSC subpopulations in vivo, with complete ablation of leukaemia. In a minority of cases, leukaemia burden was reduced following PTL treatment but not eliminated. Therefore, it may be necessary to combine PTL with other agents to improve killing of all LSC subpopulations. Another pathway of increasing interest in the treatment of leukaemias is the BCL-2 family. BCL-2 has been shown to be overexpressed in over 66% of B-ALL cases and is associated with tumourigenesis in several cancers. ABT-263 is an inhibitor of BCL-2, BCL-xL and BCL-w, it has been shown to selectively target AML LSC and is in early clinical trials in lymphoid malignancies. ABT-199 is another promising inhibitor that is currently in clinical trials for CLL. ABT-199 is specific for BCL-2 and has minimal risk for thrombocytopenia. In the present study the effects of both ABT-263 and ABT-199 alone or in combination with PTL were assessed in childhood ALL samples to determine whether toxicity to leukaemia cells could be improved in vitro and in vivo. The viability of bulk cells from 11 B cell precursor (BCP) ALL cases and 11 cord blood (CB) samples following drug treatment for 24 hours were assessed using flow cytometry by staining with Annexin V and propidium iodide. Initially, PTL was used at a range of 1 to 10μM, ABT-263 from 0.025 to 1μM and ABT-199 from 0.1 to 10μM. Only PTL and ABT-263 significantly reduced the viability of ALL cells compared to CB with IC50 values of 1.2μM and 0.125μM (P≤0.01 and P≤0.0015), respectively. In vitro drug combination studies demonstrated synergism when combining PTL with ABT-263 in a 9.5:1 ratio using the Chou Talalay model. The viability of ALL cells following combination therapy (1.2μM PTL with 0.125μM ABT-263) was reduced to 38.3±32.5%, while CB viability was unaffected (96.9±29%, P<0.0001). Using this combined dose, toxicity to ALL cells was increased by a further 35% compared to PTL alone and by 25% compared to ABT-263 alone. Even at the highest combined doses tested (9.6μM PTL: 1μM ABT-263) normal CB remained relatively unaffected with 73.3±25% surviving. The effects of these drugs alone and in combination were also assessed in LSC subpopulations in 3 of these cases. Unsorted ALL cells, CD34+/CD19+ and CD34-/CD19+ subpopulations were the most responsive with viabilities ranging from 17.6±4% to 23.9±11% using 1.2μM PTL and 0.125μM ABT-263. The CD34+/CD19- and CD34-/CD19- cells were more resistant with 70.3±40% and 73.3±15% surviving, respectively. However, since we have previously shown that the effects of in vitro drug treatment do not always accurately reflect the response in vivo, it was important to evaluate the effects of these drugs in mice with established leukaemia. NOD/LtSz-scid IL-2Rγc null (NSG) mice were inoculated with 1-1.15x106 unsorted BCP-ALL cells. Once the levels of leukaemia engraftment in murine peripheral blood reached ≥ 0.1%, mice were treated with 100mg/kg ABT-263 or ABT-199 and vehicle by oral gavage for 21 consecutive days and the levels of leukaemia burden were monitored weekly. Results to date demonstrate that leukaemia levels continued to rise in placebo-treated mice, reaching 49.2±7% by day 21, while the levels in ABT-263 and ABT-199 treated mice were significantly lower at 8.6±10% and 23.7±12%, respectively (P<0.0001). The use of ABT-263 and ABT-199 significantly improved the survival of NSG compared to untreated controls (P=0.0001). These data indicate that combining PTL with ABT-263 shows promising results in the killing of bulk and LSC populations in BCP-ALL. Ongoing in vivo studies will assess the potential of using BCL-2 inhibitors in combination with PTL compared to standard chemotherapeutics. Disclosures No relevant conflicts of interest to declare.

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Effect of Parthenolide on Stem Cells in Childhood Acute Lymphoblastic Leukaemia.

Blood ◽

10.1182/blood.v112.11.1341.1341 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1341-1341

Author(s):

Paraskevi Diamanti ◽

Charlotte V Cox ◽

Allison Blair

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Acute Lymphoblastic Leukaemia ◽

Lymphoblastic Leukaemia ◽

Therapeutic Agents ◽

Stem Cell Population ◽

Normal Peripheral Blood ◽

Childhood Acute Lymphoblastic Leukaemia ◽

Short Term Exposure

Abstract Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 80% in recent years. Despite these successes, around 15% of patients die of the disease and relapse is the most common cause of treatment failure. Intensification of treatment to prevent or treat relapse may not be a feasible approach due to an increased risk of significant adverse effects. It is possible that ALL may be maintained by a subpopulation of stem cells that are resistant to regimens designed to kill the bulk population and subsequent relapses may arise from these stem cells. Consequently, there is a need to assess the efficacy of therapeutic agents on ALL stem cells. We have previously shown that ALL cells that are capable of initiating and sustaining the disease in serial xenografts have a CD34+/CD19− phenotype. Furthermore, these putative ALL stem cells were resistant to treatment in vitro with dexamethasone and vincristine, two agents routinely used in the treatment of paediatric leukaemia. In this investigation we have examined the effects of the sesquiterpene lactone parthenolide (PTL), a natural compound which induces oxidative stress and inhibits NF-kB. PTL effectively eradicates stem cells in AML and B-CLL in vitro while sparing normal heamopoietic cells. Unsorted leukaemia cells from 11 cases, with mixed prognostic subgroups, were co-cultured with and without PTL at a range of 0–10μM for 18–24 hours. Cell viability and apoptosis were evaluated by flow cytometry using annexin V and propidium iodide staining. Four out of the 11 cases were relatively resistant to treatment with PTL with only small reductions in viability (<5%) and no significant effect on apoptosis, even at the highest dose evaluated. There was no correlation between the prognostic risk group and the response to PTL. Primary cells from the 4 resistant cases were sorted into CD34+/CD19+ and CD34+/CD19− subfractions to assess the effect of PTL on these cells. The effects of PTL on the CD34+/CD19+ population were similar to that observed with the unsorted leukaemia cells. The CD34+/CD19− population was completely resistant to treatment with PTL, with more cells surviving treatment than the unsorted cells (P=0.03). In the 7 responding cases, the viability of the unsorted cells decreased to 28.4±7.1% and 38±12% were apoptotic following treatment. Very similar effects were observed with the CD34+/CD19+ subfraction in these responding cases with viability reduced to 33.4±6% and 35.9±14% were apoptotic. In contrast, the CD34+/CD19− cells from these 7 cases were significantly resistant to PTL with viabilities >75% at all concentrations evaluated (P<0.003). Apoptosis was 2.6-fold lower at 10μM PTL in the CD34+/CD19− subfraction compared to the unsorted cells (P=0.05). FISH analyses were performed on the viable cells at the end of the time-course and confirmed that leukaemia cells were surviving treatment with PTL. CD34+/CD38− cells from normal peripheral blood samples were also found to be resistant to treatment with PTL and survival of these cells at 10μM was not significantly different to that observed in the CD34+/CD19− ALL population in all 11 cases (P=0.38). Studies to assess the functional capacity of PTL-treated ALL cells are ongoing. These data demonstrate that while PTL shows promising effects on the bulk leukaemia population in some ALL cases, it had no significant effect on the putative ALL stem cell population. In each case examined, the CD34+/CD19− cells were resistant to short term exposure to PTL and responded in a similar manner to normal haemopoietic stem cells. These findings highlight the importance of evaluating therapeutic agents in the context of leukaemia stem cell populations and not just on the bulk leukaemia. Future studies are warranted to gain insight into how the drug sensitivity of ALL stem cells may be mediated.

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Myeloid antigen co-expression in childhood acute lymphoblastic leukaemia: relationship with in vitro drug resistance

British Journal of Haematology ◽

10.1046/j.1365-2141.1999.01440.x ◽

1999 ◽

Vol 105 (4) ◽

pp. 876-882 ◽

Cited By ~ 19

Author(s):

M. L. Den Boer ◽

P. Kapaun ◽

R. Pieters ◽

K. M. Kazemier ◽

G. E. Janka-Schaub ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukaemia ◽

Lymphoblastic Leukaemia ◽

Childhood Acute Lymphoblastic Leukaemia

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A Novel Combination Therapy for Paediatric T Cell Acute Lymphoblastic Leukaemia

Blood ◽

10.1182/blood.v126.23.3767.3767 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3767-3767

Author(s):

Ben Christopher Ede ◽

Paraskevi Diamanti ◽

Charlotte V. Cox ◽

Allison Blair

Keyword(s):

Gene Expression ◽

T Cell ◽

Combination Therapy ◽

Acute Lymphoblastic Leukaemia ◽

Lymphoblastic Leukaemia ◽

Nsg Mice ◽

Gene And Protein Expression ◽

Apoptotic Proteins

Abstract T cell acute lymphoblastic leukaemia (T-ALL) is a rare form of leukaemia that accounts for approximately 15% of paediatric ALL cases. Unfortunately, approximately 20% of patients do not achieve long term remission as a result of failure of therapy to eradicate the disease. T-ALL is a highly heterogeneous disease that displays a spectrum of immunophenotypes, chromosomal aberrations and gene expression profiles. This heterogeneity has prompted research into more targeted therapies, with the aim of overcoming drug resistance often found with standard chemotherapeutic regimens. Here, we build upon use of the drug Parthenolide (PTL), which has shown promise in treatment of T-ALL and other leukaemias such as BCP-ALL and AML, in combination with ABT-263, a BCL-2 family antagonising agent. Bone marrow samples from 10 T-ALL cases, taken at diagnosis, were treated with PTL in vitro for 24 hours then viability was assessed using the annexin V / PI flow cytometric assay. Variable cytotoxic effects were observed in samples treated with PTL (1-10µM), with half maximal inhibitory concentrations ranging from 2.6-10 µM. At the highest dose tested, the proportion of surviving cells ranged from 5.79-56% (median 35.33%). BM from 5 of these samples was used for whole genome microarray (WGA) analysis. We compared gene expression in bulk ALL and in specific subpopulations, known to have leukaemia initiating capacity in vivo; CD34+/CD7+, CD34+/CD7-, CD34-/CD7+ and CD34-/CD7- cells. WGA data demonstrated that CD34+/CD7- was the only subpopulation to express significantly lower levels (5.38 fold) of the pro-apoptotic gene Bcl-2L11 (BIM) compared to the unsorted bulk T-ALL cells, p=0.006. Interestingly, we have previously shown that CD34+/CD7- cells from a few patients were resistant to PTL treatment in vivo compared to unsorted cells. To validate these results, mRNA and relative protein quantification was performed by qPCR and western blotting in bulk material from 8 of the 10 samples, 3 of which had been analysed by microarray for BIM expression. We found that the gene and protein expression levels of BIM were negatively correlated with PTL resistance in vitro, p≤0.0001 and p=0.049 respectively. This suggests that reduced BIM expression is related to PTL resistance. We next evaluated the effects of combining PTL and ABT-263 on T-ALL cells in vitro. ABT-263 is a BH3 protein mimetic, like BIM it promotes apoptosis by blocking the inhibitory effects that BCL-2 anti-apoptotic proteins have on pro-apoptotic proteins. The effects of combining the drugs were assessed in 7 of the original 10 samples. Unsorted ALL cells were incubated with PTL and ABT-263 for 24 hours, before viability was analysed by flow cytometry and drug synergy was calculated via the Chou Talalay method. This drug combination showed enhanced cytotoxicity to T-ALL cells compared to PTL (p=0.0282) or ABT-263 (p=0.0358) alone. Moreover, the highest combined dose tested (2.5µM PTL with 0.25µM ABT-263) killed 86.1±9% cells cf 71.8±18% with ABT-263 alone and only 21.7±11% with PTL alone. The combination also showed synergism with a combination index value below 1 in all doses tested. Previous findings in our laboratory have shown that in vivo PTL treatment eliminated childhood leukaemia in NOD/LtSz-scid IL-2Rγc null (NSG) mice, in most cases tested. It may be possible to further enhance this toxicity using ABT-263 alone or in combination with PTL. NSG mice were inoculated with unsorted T-ALL cells and leukaemia was allowed to establish until levels in peripheral blood (PB) exceeded 0.1%. NSG mice were subsequently treated orally for 21 days with 100mg/kg of ABT-263 or placebo and leukaemia burden was monitored weekly in PB aspirates. Twenty-eight days following commencement of treatment, leukaemia burden in the placebo treated group was 80.73±2.94% and the animals were electively culled. In contrast, disease burden was significantly lower in the treated animals at this stage (35.2±2.1%, p=0.004). ABT-263 treatment has significantly improved survival of all xenografts to date, (P<0.014). In summary, we have shown that PTL resistance is related to the expression of BIM. By combining PTL with ABT-263, which mimics the pro-apoptotic action of BIM, the drugs work synergistically to enhance T-ALL cytotoxicity in vitro. Ongoing in vivo studies will assess the full potential of this combination therapy for paediatric T-ALL. Disclosures No relevant conflicts of interest to declare.

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