CML In Mice Research Articles

Novel Dicarboximide BK124.1 Breaks Multidrug Resistance and Shows Anticancer Efficacy in Chronic Myeloid Leukemia Preclinical Models and Patients' CD34+/CD38- Leukemia Stem Cells.

Simple SummaryChemotherapy is a first line treatment in many cancer types, but the constant exposition to chemotherapeutics often leads to therapy resistance. An example is chronic myeloid leukemia that, due to the use of tyrosine kinase inhibitors such as imatinib, remains manageable, however incurable. Overall, 20–25% of imatinib responders develop secondary resistance, and among them, 20–40% is due to mechanisms such as expression of P-glycoprotein (MDR1) or leukemia stem cells’ mechanisms of survival and cancer regrowth. This study provides the first evidence from animal and cellular models that this resistance can be overcome with the novel dicarboximide BK124.1. The compound causes no visible toxicity in mice, and has proper pharmacokinetics for therapeutic applications. It was efficient against both multidrug resistant CML blasts and CD34+/CD38− leukemia stem cells coming from CML patients. Future development of BK124.1 could offer curative treatment of CML and of other cancers resistant or intolerant to current chemotherapy.The search is ongoing for new anticancer therapeutics that would overcome resistance to chemotherapy. This includes chronic myeloid leukemia, particularly suitable for the studies of novel anticancer compounds due to its homogenous and well-known genetic background. Here we show anticancer efficacy of novel dicarboximide denoted BK124.1 (C31H37ClN2O4) in a mouse CML xenograft model and in vitro in two types of chemoresistant CML cells: MDR1 blasts and in CD34+ patients’ stem cells (N = 8) using immunoblotting and flow cytometry. Intraperitoneal administration of BK124.1 showed anti-CML efficacy in the xenograft mouse model (N = 6) comparable to the commonly used imatinib and hydroxyurea. In K562 blasts, BK124.1 decreased the protein levels of BCR-ABL1 kinase and its downstream effectors, resulting in G2/M cell cycle arrest and apoptosis associated with FOXO3a/p21waf1/cip1 upregulation in the nucleus. Additionally, BK124.1 evoked massive apoptosis in multidrug resistant K562-MDR1 cells (IC50 = 2.16 μM), in CD34+ cells from CML patients (IC50 = 1.5 µM), and in the CD34+/CD38− subpopulation consisting of rare, drug-resistant cancer initiating stem cells. Given the advantages of BK124.1 as a potential chemotherapeutic and its unique ability to overcome BCR-ABL1 dependent and independent multidrug resistance mechanisms, future development of BK124.1 could offer a cure for CML and other cancers resistant to present drugs.

Gadd45 in Normal Hematopoiesis and Leukemia.

Gadd45a, Gadd45b, and Gadd45g have been implicated in cell cycle arrest, DNA repair, apoptosis, innate immunity, genomic stability, and modulation of normal blood cell development and leukemia. Each of the Gadd45 genes was shown to be regulated independently in myeloid cells in response to cytokine stimulation modulating blood cell survival and differentiation, including maintaining the quiescent stem cell pool. Gadd45a and Gadd45b were also shown to mediate the protective effects from UV in hematopoietic cells by separate signaling pathways involving either p38 activation or JNK inhibition. Furthermore, it was shown that gadd45a methylation in AML is predictive of poor survival. It was also shown that loss of Gadd45b accelerates the development of BCR-ABL driven CML in mice and leads to decreased median survival. The Gadd45b-deficient CML progenitors exhibited increased proliferation and decreased apoptosis, and this was associated with hyper-activation of c-Jun NH2-terminal kinase and Stat5. Moreover, loss of Gadd45a also accelerated the development of BCR-ABL driven CML, and this was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. In human patients with chronic phase CML, gadd45a expression is up-regulated, whereas in accelerated and blast crisis phase patients, gadd45a is downregulated. Collectively, these results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may serve as a unique prognostic marker of CML progression. Thus Gadd45 proteins provide excellent targets for leukemia therapy.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed.The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs).Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells.To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner.The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive.Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure).To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table).We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. [Display omitted] [Display omitted] DisclosuresMichaels:Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

BACH2 promotes Lineage-Specific Fate Decisions in BCR-ABL1-Driven Leukemia

Background and Hypothesis: CML and Ph+ ALL are both driven by the oncogenic BCR-ABL1 tyrosine kinase and CML can progress into lymphoid blast crisis (LBC), which is clinically and biologically indistinguishable from Ph+ ALL. We hypothesize that the stark differences in phenotype and clinical outcomes between CML and Ph+ ALL/LBC are due to lineage-specific transcription factors. Our group recently identified BACH2 as a B cell-specific transcription factor that mediates negative selection at the pre-B cell receptor checkpoint and functions as a tumor suppressor in Ph+ ALL and LBC (Swaminathan et al., Nature Med 2013). Here we report the surprising finding that BACH2 mediates lineage-specific fate decisions in BCR-ABL1 driven leukemia: while a potent tumor suppressor in B cell lineage in Ph+ ALL and LBC, BACH2 is required for survival and self-renewal of myeloid lineage CML cells.Results: A gene expression analysis in 99 patients with CML (Radich et al, 2006) revealed that patients with higher blast counts showed significantly higher gene expression values of BACH2 (P=8.88 x 10-9), suggesting that BACH2 mRNA levels increase with CML progression. To determine the mechanistic role of BACH2 in the progression of CML, we studied genetic deletion of BACH2 in a CML mouse model. To this end, we transformed Lin-kit+Sca-1+(LSK) cells from Bach2+/+ and Bach2-/- bone marrow with BCR-ABL1. We compared these cells alongside with Bach2+/+ and Bach2-/- pre-B cells that were transformed with BCR-ABL1 as a model for Ph+ ALL and LBC. Bach2+/+ and Bach2-/- CML-like and Ph+ ALL/LBC-like leukemia cells were then tested in a series of functional experiments to test their ability to initiate fatal leukemia in transplant recipients. While deletion of BACH2 accelerated B cell lineage leukemia (Ph+ ALL/LBC-like) in transplant recipients, we noted the opposite outcome in transplant experiments with myeloid lineage CML cells: All mice receiving Bach2+/+BCR-ABL1-transformed LSK cells developed CML-like disease within 60 days whereas recipients of Bach2-/- cells did not develop CML-like disease (Bach2+/+ vs Bach2-/-, median survival time= 33 days vs undefined; P=0.001). Additionally, we observed that deletion of Bach2 loss reduced both the colony formation ability (P=0.0059) and the S phase proliferation potential (P=0.0075) of the CML progenitors. These results were in contrast to those observed in Ph+Bach2+/+ and Bach2-/- ALLs. Reconstitution of Bach2 in Bach2-/- CML cells rescued its colony forming ability (P= 0.023). These findings were confirmed in primary human CML cells from two patients with CML in chronic phase: Inducible overexpression of Bach2 in patient-derived CML cells conferred a selective proliferative advantage of BACH2 overexpressing cells over time, in comparison to empty vector controls. Bach2 triggered a survival program in human and mouse CML cells, in contrast to B cell lineage Ph+ ALL and LBC cells. Interestingly, Bach2 did not provide a selective growth advantage to untransformed myeloid and multi-lineage progenitor cells. This demonstrates that Bach2 drives lineage-specific fate decisions and cooperates with the transforming BCR-ABL1 oncogene. Utilizing an in vitro lineage-switch assay overexpressing CEBPα we characterized the opposing roles of BACH2 in Ph+ ALL and CML. Cell viability and colony formation assays demonstrated that absence of Bach2 results in increased survival and proliferation of Ph+ ALL cells. In striking contrast, however, CEBPα-induced myeloid reprogramming induced cell cycle arrest and suppressed colony formation in Bach2-deficient cells (P=0.0001).Conclusion: While BACH2 is primarily expressed in the B cell lineage, functioning as a tumor suppressor in Ph+ ALL, we report here the surprising finding that BACH2 is required for survival and self-renewal of myeloid lineage CML cells. BACH2 mediates lineage-specific fate decisions in BCR-ABL1 driven leukemia and represents one example of how conversion of lineage identity (e.g. from CML to LBC) may impact specific requirement for malignant transformation. For instance, deletions of BACH2 at 6q15 are common in CML at the time of LBC progression but are not found in CML chronic phase or myeloid blast crisis. DisclosuresNo relevant conflicts of interest to declare.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

AbstractAbstract 3391Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a.We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010).Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures:No relevant conflicts of interest to declare.

Abstract 4237: Foxo3a is essential for survival of leukemia-initiating cells in chronic myeloid leukemia

Abstract Chronic myelogenous leukemia (CML) is caused by a defined genetic abnormality, BCR-ABL, a constitutively active protein tyrosine kinase. Although the introduction of imatinib, a small molecule inhibitor of ABL, represented a breakthrough in the treatment of CML, major part of patients treated in chronic-phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor of the production of differentiated leukemic cells, but does not deplete leukemia-initiating cells (LICs). To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, we here evaluated molecular mechanisms on survival of the CML LICs. We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin-Sca-1+ (KLS+) population in BCR-ABL+ CML cells, as previously reported. Unexpectedly, phosphorylation levels of Akt in CML LICs appeared to be lower than that in non-LICs, KLS- cells, despite it is widely believed that BCR-ABL induces activation of Akt signal. Since Forkhead O (Foxo) transcription factors, which are important downstream targets of PI3K-Akt signaling, are essential for the maintenance of self-renewal capacity of normal HSCs, we focused on analysis of Foxo3a in CML. Consistent with Akt phosphorylation status, we found that cells with nuclear localization of Foxo3a were enriched in the CML LICs, whereas non-LICs showed the cytoplasmic localization, suggesting that Foxo3a is activated via Akt inactivation in the CML LICs, but not in majority of CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at 3rd transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at 3rd transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-3rd transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. Furthermore, deficiency of Foxo3a led to enhanced efficiency in elimination of CML LICs in combination with imatinib treatment. These results demonstrate a critical role of Foxo pathway in survival of CML LICs, and provide a novel therapeutics approach for CML patients by suppression of FOXO signaling. 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 4237.

BCL6 Is Required for Leukemia-Initiation and Self-Renewal Signaling in Chronic Myeloid Leukemia.

Abstract 2167Poster Board II-144The transcriptional repressor BCL6 was discovered as a potent protooncogene in diffuse large B-cell lymphoma (DLBCL) and is also expressed in normal germinal center (GC) B cells. BCL6 protects DLBCL and GC B cells against DNA damage-induced apoptosis by suppressing target genes including p53, p21, ATR and p27. In preliminary experiments for this study, we found that treatment of BCR-ABL1-driven Ph+ ALL cells with Imatinib results in a 60- to 90-fold up-regulation of BCL6, reaching protein levels as high as in DLBCL and GC B cells. Since the oncogenic BCR-ABL1 kinase also drives leukemic transformation in CML cells, we studied regulation of BCL6 in human CML cells upon TKI-treatment. Like in Ph+ ALL, TKI-treatment resulted in rapid BCL6 mRNA and protein upregulation in CML cells, albeit to lesser degree than in Ph+ ALL and DLBCL. We hypothesized that upregulation of BCL6 in CML cells in response to TKI-treatment serves the same function as in DLBCL and protects from p53-mediated apoptosis. Through transcriptional repression of p53, p21, ATR and p27 as in DLBCL, upregulation of BCL6 in response to TKI-treatment would represent a defense mechanism of CML cells to evade TKI-induced cell death.We tested the role of BCL6 in CML in a genetic loss-of-function experiment: To this end, myeloid progenitor cells from bone marrow of BCL6+/+ and BCL6-/- mice were transformed with BCR-ABL1. Compared to their BCL6+/+ counterparts, BCL6-/- mouse CML cells were highly sensitive to Imatinib-treatment. In a complementary approach, we treated human CML cells with either Imatinib alone or with a novel BCL6 peptide inhibitor (Retro-inverso BCL6-peptide inhibitor, RI-BPI). Consistent with findings in BCL6-/- mouse CML cells, also human CML cells were significantly sensitized to Imatinib by BCL6 inhibition (using 5 mmol/l RI-BPI). Of note, the BCL6 inhibitory peptide RI-BPI alone induced no significant toxicity in human CML cells but synergized with Imatinib-treatment.Since RI-BPI alone had no cytotoxic effect on CML cells, we tested whether loss of BCL6 function affects self-renewal capacity of CML cells. This possibility was explored in two complementary colony formation assays: In one set of experiments, we tested the ability of BCL6+/+ and BCL6-/- mouse CML cells to form colonies in semisolid methylcellulose agar. The comparison between BCL6+/+ (94 ± 11 colonies) and BCL6-/- (<1 ± 1 colonies; p<0.004) mouse CML cells revealed that BCL6 represents an absolute requirement for self-renewal signaling in CML cells. In line with these results, we observed that BCL6-/- mouse CML cells undergo replicative senescence and cell cycle arrest under cell culture conditions after ∼2 months, whereas leukemic growth proceeds undiminished in BCL6+/+ CML cells. These results were confirmed studying BCL6 function in human CML cells using RI-BPI. Despite lack of toxicity, 5 mmol/l RI-BPI drastically reduced the number of colonies that were formed by human CML cells (980 ± 79 vs 262 ± 174; p= 0.001). Based on these findings we conclude that inhibition of BCL6 using a novel peptide inhibitor effectively disrupts self-renewal signaling and may be developed as a novel therapeutic approach for leukemia stem cell eradication in CML. Disclosures:No relevant conflicts of interest to declare.

6 Molecular biology and leukaemia diagnosis

The diagnosis and classification of leukaemia started with simple morphological examination and now embraces use of special stains, cytochemistry and immunophenotyping. Genetic studies have progressed from karyotyping to detection of genetic changes within genes. The methods described in this chapter are still at an early stage of development and, so far, have provided relatively little in the way of an extension of available diagnostic information. Sometimes the methods provide extensions to existing techniques, for example by the detection of bcr rearrangements in patients who have CML or ALL but do not have a detectable Philadelphia chromosome. Another example is retrospective diagnosis of gene rearrangements using DNA from slide preparations. However, it should be noted that it has only very recently been shown that there is likely to be a causal relationship between the Ph chromosome and leukaemia. Daley et al (1990) induced CML in mice by bone marrow transplantation of cells infected with a retrovirus encoding P210bcr/abl and Heisterkamp et al (1990) produced mice transgenic for a BCR/ABL P190 DNA construct and showed that the progeny died of acute leukaemia (mostly ALL). We have not summarized studies of the incidence of activated oncogenes such as RAS in leukaemia and myelodysplasia. Such oncogenes appear to be involved in many tumours and may well indicate either a predisposition to cancer or a particular stage of malignancy, but their analysis does not at present help in making a diagnosis. It is likely that, as we understand more about the nature of the malignant process, we shall be able to use genetic techniques to enhance considerably both diagnostic and prognostic precision.