ALBERT

Description: Abstract 202 Background: Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months. Approach: Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53. BCL6-Null CML cells fail to initiate leukemia: Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by 〉15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a 〉12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by 〉7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (〉400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032), Conclusion: Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Disclosures: Hochhaus: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 1724 Poster Board I-750 The success of imatinib (IM, formerly STI571, Gleevec®) in the treatment of chronic myeloid leukemia (CML) is compromised by the development of primary or acquired IM-resistance, particularly in advanced phase disease as well as by a limited IM-effect on immature hematopoietic stem cells, emphasizing the need for novel therapeutic strategies. The small molecule inhibitor Danusertib (formerly PHA-739358) potently inhibits Aurora and ABL kinases. Here, the individual contribution of each pathway to the effect of Danusertib was investigated. Starting at very low concentration, a dose-dependent reduction of BCR-ABL activity was observed, whereas inhibition of Aurora kinase activity, assessed by phosphorylation of histone H3-Ser10, required substantially higher concentrations. In primary CD34+ CML cells, including initially quiescent leukemic stem cells, combination therapy with IM and Danusertib revealed a synergistic anti-proliferative activity, which also affected immature CD34+38- cells. Neither mono- nor combination therapy led to substantial induction of apoptosis in quiescent stem cells. Interestingly, under treatment with Danusertib, the emergence of resistant clones in a well-established murine Ba/F3-p210 cell model was considerably less frequent than with IM. Surprisingly, Danusertib-resistant cells did not have mutations in BCR-ABL or Aurora kinase domains and remained IM-sensitive. Analysis of resistance mechanisms using DNA-microarray suggests an overexpression of Abcg2 efflux transporter to be causative for the resistance arising under Danusertib treatment. In support of this finding, stable retroviral overexpression of Abcg2 in parental Ba/F3-p210 cells induced a resistant phenotype against Danusertib. Furthermore, the Abcg2 inhibitor Fumitremorgin C (FTC) could restore the sensitivity of resistant cells to Danusertib. Finally, significant re-expression of Abcg2 in parental Ba/F3-p210 cells upon treatment with the demethylating agent 5-Azacytidine suggests that an epigenetic mechanism might play a role in the regulation of Abcg2 gene expression in resistant clones. Detailed analyses of the methylation patterns of the Abcg2 promoter region are currently being performed. In conclusion, simultaneous in vitro exposure of Ba/F3-p210 cells to Danusertib and IM significantly reduced the emergence of drug resistance, raising hope that both epigenetic modulation of drug transporters involved in development of resistance as well as hypothesis-driven combinations of kinase inhibitors may eventually achieve durable disease control even in 2nd and 3rd line treatment of CML. Disclosures Brummendorf: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Moll:Nerviano MS: Employment. Jost:MSD: Research Funding. Bokemeyer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holyoake:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Balabanov:Novartis : Research Funding; Bristol Myers Squibb: Research Funding.

Description: Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC) disease caused by the reciprocal translocation t(9;22). Although there is clear evidence that the resulting oncogenic tyrosine kinase BCR-ABL is the key event of leukemia initiation which drives stem cell proliferation and expansion of myeloid progenitors in early chronic phase (CP), the mechanism leading to advanced phases remains elusive. Recently, we could show that telomere attrition correlates with disease stages due to increased leukemic stem cell turnover. Here, we could provide first time evidence that this can functionally contribute to disease progression in CML. In our study we made use of the well-described telomerase knockout mouse model (mTR-/-), lacking the RNA subunit of telomerase and resulting in significant telomere shortening with each generation, and retrovirally introduced BCR-ABL into primary bone marrow cells of different generation. Although all CML-like cultures (hereafter referred to as “CML”) grew exponentially and growth factor independently in vitro, they showed remarkable differences in cellular growth kinetics depending on the generation of mTR-/-mice the cells were derived from. CML-HSCs of generation iG4 (CML-iG4) are functionally impaired with respect to their growth properties and ceased to proliferate due to a robust senescent-like cell cycle arrest. Interestingly, they did not show overt genomic instability, but and are less susceptible to Imatinib-induced apoptosis compared to wildtype cells (CML-WT). In sharp contrast, CML-G2 cells with only pre-shortened telomere lengths grew most rapidly and presented with an impressive proliferation advantage compared to CML-WT and -iG4 cells, while they still retain Imatinib sensitivity. Notably, we uncovered that this growth advantage is related to a “telomere-associated secretory phenotype” (TASP), comprising the upregulation and secretion of chemokines, interleukins and other growth factors, thereby potentiating oncogene-driven growth in an autocrine fashion. In line with those observations, we found that conditioned supernatant of CML-G2 cells markedly enhanced proliferation of CML-WT and pre-senescent CML-iG4 HSCs. To investigate if a TPE (telomere position effect)-related mechanism is responsible for inducing inflammatory gene expression in BCR-ABL positive cells, we mapped selected TASP genes for their chromosomal location. However, although they are frequently found in well-known cluster (e.g. chemokines), TASP genes are not preferentially located close to the (sub-) telomere. This suggests that a yet unknown mechanism controls TASP gene expression upon telomere shortening. Most importantly, a similar inflammatory mRNA expression pattern was found in CML patients of accelerated phase (AP), but not in blast crisis (BC). Taken together, those data support the hypothesis that accelerated telomere shortening contributes to disease progression in BCR-ABL-driven leukemogenesis by the expression of an inflammatory signature, while telomere-induced senescence needs to be bypassed (e.g. by upregulation of telomerase) in order for leukemic cells to be able to progress to blast crisis (BC) CML. Disclosures: Brümmendorf: Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ariad: Consultancy.

Description: The tyrosine kinase inhibitor (TKI) Imatinib (IM) represents the gold standard firstline treatment for patients with newly diagnosed chronic myeloid leukemia (CML). For patients developing resistance or intolerance to Imatinib, the 2nd generation TKIs Dasatinib (DASA) and Nilotinib (NILO) which are approved, and Bosutinib (BOSU) which is in clinical development, possess activity against almost all mutant forms of BCR-ABL which confer Imatinib-resistance, except the gatekeeper mutation, T315I. This latter mutation occurs in approximately 15% of clinically observed mutations in chronic phase CML and confers resistance to all currently approved agents. Therefore compounds have been evaluated for activity against T315I mutant BCR-ABL, among which combined Aurora kinase and Abl inhibitors such as PHA-739358 (PHA) have been identified as showing some promise.In the current study, we used a classical proteomics approach to generate drug profiles of the TKIs (IM, DASA, NILO and PHA), in order to identify biomarkers that are either compound or drug group (i.e. 1st, 2nd or 3rd generation TKI) specific and could potentially be used as biomarkers for response prediction in vivo. For in vitro screening, we used murine Ba/F3 cells expressing wild-type (p210, wt) BCRABL or mutants, which are either low grade (M351T) or absolutely resistant (T315I) to Imatinib. Using 2D-gel electropheresis and mass spectrometry, we could identify a total of 68 individual protein spots which were differentially regulated in cells when treated with equieffective concentrations (IC50) of TKIs. Using in silico overlay of the different 2D-gels (Delta2D, Decodon GmbH Greifswald), 42, 38, 41 and 15 spots were found to be specifically differentially regulated in Ba/F3 cells expressing wt BCR-ABL under either IM, NILO, DASA and PHA, respectively. Interestingly, hierarchical cluster analysis based on these candidate proteins identified similar protein expression patterns for IM, NILO and DASA in comparison to PHA. Using genontology analysis (Panther software), the majority of the proteins belonged to the group of nucleic acid binding proteins (25%), cytoskeletal proteins (13%) and chaperones (12%). In contrast to the broad response of the different TKIs on wt Bcr-Abl cells, changes in protein expression patterns induced in cells carrying the M315T BCR-Abl mutation were substantially less pronounced (IM: 9, NILO: 12, DASA: 28, PHA: 17) with the strongest response seen in Dasatinibtreated cells, consistant with the compound being a powerful inhibitor of both wt and M351T BCR-ABL signaling. With the exception of the combined BCR-ABL and Aurorakinase inhibitor PHA (7 proteins), cells expressing T315I BCR-ABL exhibited no altered protein expression in response to treatment with either IM or the other 2nd generation TKIs. The protein expression patterns identified were used for systems biology network analysis using Metacore software (GeneGo), which enabled the elucidation of signaling pathways and identification of transcription factors involved in TKI response. Besides known regulators of BCR-ABL signaling, such as c-Myc and p53, we were able to identify novel TKI-dependent candidate proteins (e.g. eIF5a) and post-translational modifications (PTM) that, pending validation in primary patient material might effectively be used as biomarkers for response prediction in the near future.

Description: As a barrier against malignant transformation, checkpoint-mediated failsafe mechanisms such as apoptosis or cellular senescence may be activated in response to mitogenic oncogenes. Acute induction of oncogenic Ras has been shown to provoke a senescence-like cell-cycle arrest that involves the retinoblastoma (Rb) pathway. Recently, Rb-mediated formation of heterochromatin was identified as a critical feature of cellular senescence. Since methylation of histone H3 lysine 9 by the Rb-bound histone methyltransferase Suv39h1 contributes to the transcriptional repression of growth promoting genes, we asked whether inactivation of Suv39h1 may disable cellular senescence as a suppressor mechanism against malignant transformation by oncogenic Ras. To investigate the role of Suv39h1 in Ras-driven lymphomagenesis, Eμ-N-ras-transgenic mice were crossbred to mice harboring targeted deletions in the Suv39h1 locus, as well as to p53 knockout mice. Ras-transgenic mice with no additional defined genetic lesion - hereafter referred to as controls - developed a terminal condition mostly due to a neoplastic histiocytic infiltration of the liver with a median latency of about 250 days. In stark contrast, ras-transgenic mice lacking Suv39h1, or carrying heterozygous defects in the Suv39h1 or p53 locus, respectively, entered a final disease stage significantly earlier, i.e. at a median age of 50 to 100 days, due to aggressive T cell lymphomas that were virtually absent or only sporadically found in the control group. Importantly, similar to p53+/− mice which typically lost the remaining wild-type allele in lymphoma cells, Suv39h1 heterozygous mice produced tumors that invariably lacked Suv39h1 expression, rendering these lymphomas in fact Suv39h1 null. Suv39h1 null cells propagated as primary cultures grew readily in an exponential fashion, surpassed only by the growth potential of p53 null cells. In short-term cytotoxicity assays, the anticancer agent adriamycin efficiently killed both control and Suv39h1 null lymphoma cells, whereas p53 null cells were resistant, indicating no overt apoptotic defect in Suv39h1 null cells. While non-neoplastic ras-transgenic Suv39h1+/+ cells exhibited heterochromatin foci reminiscent of cellular senescence, this phenotype was completely lost in Suv39h1 null lymphoma cells. In the presence of Bcl2 - introduced to block apoptotic cell death - adriamycin-treated control cells entered cellular senescence, whereas Suv39h1 null cells failed to display a senescent phenotype in response to therapy, demonstrating that stress-inducible senescence is a Suv39h1-dependent program. The data unveil the fundamental role of cellular senescence as a tumor suppressor principle by establishing a novel link between Ras-driven transformation and deregulated histone modification in vivo. Impaired cellular senescence due to inactivation of the histone H3 lysine 9 methyltransferase Suv39h1 promotes malignant transformation by oncogenic Ras, leading to a dramatic acceleration of Ras-driven lymphoma formation. Moreover, the observation that a full-blown malignancy may arise as a consequence of disabled senescence but without an apparent apoptotic defect has important therapeutic implications.

Description: Telomeres consist of repeat structures such as (TTAGGG)n in vertebrates and are localized at the end of chromosomes. Replication-dependent telomere shortening due to the end-replication problem can be counteracted by upregulation of an endogenous reverse transcriptase called telomerase. Increasing evidence suggests that critical telomere shortening results in genetic instability which may promote tumour evolution and telomerase activation during which critically short telomeres are stabilised and ongoing tumour growth is facilitated. In Chronic myeloid leukemia (CML) the high turnover of the malignant clone is driven by the oncogene BCR-ABL and leads to accelerated telomere shortening in chronic phase (CP) compared to telomere length in healthy individuals. Telomere shortening has been demonstrated to be correlated with disease stage, duration, prognosis and response to molecular targeted treatment. Despite of the accelerated telomere shortening observed, telomerase activity is increased in CP CML and further upregulated with progression of the disease to accelerated phase or blast crisis (AP/BC). To investigate the effect of telomerase inhibition on BCR-ABL-positive cells, we expressed a dominant-negative mutant of hTERT (vector pOS DNhTERT-IRES-GFP) in K562 cells. The cells were single sorted and clones in addition to bulk cultures were long term expanded in vitro. The expression of the transgene DNhTERT was monitored by the expression of GFP and function of DNhTERT was analyzed by measurement of telomere length (by flow-FISH) and telomerase activity (TRAP assay). Evaluation of these parameters showed the following patterns of growth kinetics and telomere biology in individual clones: Two clones lost telomere repeats and were transiently delayed in growth kinetics but eventually escaped from crisis without loss of GFP expression (indicated by a re-increase in telomere length and growth rate, group A) Three other clones lost GFP expression after initial and significant telomere reduction indicating loss of the transgene (group B). Finally, telomere length and growth kinetics of two remaining clones and of the bulk culture cells remained unaffected by expression of DN-hTERT (group C). Of note, none of the clones analyzed either died or entered cell cycle arrest. Further analyses of one clone of group A revealed impaired DNA damage response indicated by two fold increase in number of γH2AX foci in comparison to control cells. Moreover, the expression pattern of genes involved in DNA repair was significantly altered (Dual chip®). Network analysis of the altered genes using MetaCore® software confirmed p53 as a key regulator in signaling of DNA damage in these cells. CML blast crisis cell lines such as K562 are typically negative for functional p53 and p16INK4. Therefore, we went on and investigate if the presence of functional p53 is required for the induction of telomere-mediated apoptosis or senescence in BCR-ABL-positive cells. For this purpose, we restored p53 in telomerase-negative clones by using an inducible system (vector pBABE p53ERtam) in two clones from group A and group B. Induction of p53 in cells with critically short telomeres (telomere length 4–5 kb) lead to immediate induction of apoptosis while vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is dependent on the presence of functional p53 in BCR-ABL-positive cells which argues in favour of applying these strategies preferentially in CP as opposed to BC.