ALBERT

Description: Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. Three subtypes of AML carrying specific translocations, namely t(15;17), t(11;17) and t(6;9), are notable for being associated with a smaller number of co-existing driver mutations than e.g. AML with normal karyotype. This strongly suggests that the function of their aberrant gene products, PML/RAR and DEK/CAN, respectively, may subsume the functions of other driver mutations. Thus we hypothesized that these functions, while as yet elusive, not necessarily require sequential acquisition of secondary genomic alterations. We elected to study AML with the t(6;9), defined as a distinct entity by the WHO classification, because of its particular biological and high risk clinical features and unmet clinical needs. Most t(6;9)-AML patients are young, with a median age of 23-40 years, complete remission rates do not exceed 50% and median survival after diagnosis is only about 1 year. We used a novel "subtractive interaction proteomics" (SIP) approach to understand the mechanisms by which the t(6;9)-DEK/CAN nuclear oncogene induces this highly resistant leukemic phenotype. Based on Tandem Affinity Precipitation (TAP) for the enrichment of proteins complexes associated with SILAC-technology followed by LC-MS/MS we developed SIP as a comparison between the interactome of an oncogene and those of its functionally inactive mutants in order to obtain eventually only relevant interaction partners (exclusive binders) in the same genetic background. This is achieved by the subtraction of binders that are common to four functionally inactive mutants classifying them as not relevant. Bioinformatic network analysis of the 9 exclusive binders of DEK/CAN revealed by SIP (RAB1A, RAB6A, S100A7, PCBD1, Clusterin, RPS14 and 19, IDH3A, SerpinB3) using BioGrid, IntAct and String together with Ingenuity© Pathway Analysis (IPA), indicated a functional relationship with ABL1-, AKT/mTOR-, MYC- and SRC family kinases-dependent signaling. Interestingly, we found all these signaling pathways strongly activated in an autonomous manner in four DEK/CAN-positive leukemia models, DEK/CAN expressing U937 cells, t(6;9)-positive FKH-1 cells, primary syngeneic murine DEK/CAN-driven leukemias, and t(6;9)-positive patient samples. Bioinformatic analysis of the phopshoproteomic profile of FKH1 cells upon molecular targeting of single pathways (imatinib for ABL1, PP2 for SFKs, dasatinib for ABL1/SFK and Torin1 or NVP-BEZ-235 for mTOR/AKT) revealed that these signaling pathways were organized in clusters creating a network with nodes that are credible candidates for combinatorial therapeutic interventions. On the other hand inhibition of individual outputs had the potential to activate interconnected pathways in a detrimental manner with consequential clinical impact e.g. the activation of STAT5 by the inhibition of mTOR/AKT in these cells. Treatment of mice injected with primary syngeneic DEK/CAN-induced leukemic cells with dasatinib (10mg/kg) and NVP-BEZ-235 (45mg/kg) alone and in combination for 14 days led to a strong reduction of leukemia burden in all cohorts (each cohort n=7). In fact, as compared to untreated controls (146.6 +/- 36mg), mice treated with NVP-BEZ 235 alone and in combination (61.7 +/-4.7mg and 65.3+/- 4.6mg, respectively) showed a statistically significant reduction of spleen size whereas those treated with dasatinib alone (77.5 8 +/- 5.4mg) did not reach statistical significance. Taken together the here presented results reveal specific interdependencies between a nuclear oncogene and kinase driven cancer signaling pathways providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling. In addition, it provides evidence for the need of a more in depth analysis of indirect effects of molecular targeting strategies in a preclinical setting not only in AML but in all cancer types. Disclosures Ottmann: Novartis: Consultancy; Pfizer: Consultancy; Fusion Pharma: Consultancy, Research Funding; Amgen: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Incyte: Consultancy, Research Funding.

Description: The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor α (PML/RARα) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RARα, bind all-trans-retinoic acid (t-RA), and act as t-RA–dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15; 17) and expressing the PML/RARα products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RARα product has been found associated with a poorer prognosis than bcr1-PML/RARα. In the present study we have investigated the structural and functional properties of the bcr3-PML/RARα in comparison to the previously characterized bcr1-PML/RARα. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RARα APL patients and from bcr3-PML/RARα COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RARα receptor than to bcr1-PML/RARα or RARα (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RARα product but not in the bcr1-PML/RARα product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RARα isoform than to the bcr1-PML/RARα or RARα. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RARα product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the βRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RARα products can be measured.

Description: Leukemia-specific translocations - t(15;17), t(11;17), or t(8;21) - lead to the expression of leukemia associated fusion proteins (LAFP) such as PML/RAR, PLZF/RAR and AML-1/ETO. LAFP induce and maintain a leukemic phenotype by blocking terminal differentiation of hematopoietic progenitors and by increasing the self renewal potential of the leukemic stem cells (LSC). The key mechanims by which LAFP increase LSC self renewal is the activation of the Wnt-signaling pathway by up-regulating of γ-catenin and β-catenin at a transcriptional level. The aberrant activation of Wnt-signaling by the LAFP decisively contributes to the pathogenesis of AML. To disclose whether a “leukemic stem cell therapy” is effective we targeted the Wnt-signaling by Sulindac Sulfid (SuSu) in PML/RAR- or PLZF/RAR- (X-RAR) positive stem cell models. SuSu represents the active metabolite of Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), known to inactivate the Wnt-signaling. SuSu was used at a clinically achievable concentration of 50–100 μM. As leukemia models we used U937 cells stably expressing the X-RAR and NB4 cells. As stem cell models we used i.) the aldehyde dehydrogenase positive/CD34+/CD38- fraction of the KG-1 cells stably expressing the X-RAR; ii.) Sca-1+/lin- murine HSC retrovirally transduced with the X-RAR in semi solid medium. The amount of total γ-catenin and β-catenin and activated β-catenin was determined by immunoblotting. We report that SuSu i.) down-regulated not only β-catenin but also γ-catenin in X-RAR expressing U937 and KG-1 cells; ii.) reduced the active form of β-catenin in the presence of X-RAR; iii.) induced a high apoptosis rate in PML/RAR-positive NB4 cells; iv.) reduced the “stem cell fraction” of KG-1 cells expressing X-RAR but not of mock transfected controls; iv.) reduced the self renewal potential of X-RAR-positive HSC as revealed by a significantly reduced replating efficiency. Here we provide evidence that the exposure to therapeutically achievable dosages of a NSAID revert the aberrant activation of the Wnt-signaling by LAFP. The significant reduction of the aberrant self renewal potential of HSC in the presence of X-RAR further support that the inhibition of the aberrantly activated Wnt signaling in AML might be a valid molecular therapy approach which has to further validated in in vivo leukemia models and in a more clinical setting.

Description: Abstract 2424 Acute myeloid leukemia (AML) is characterized by an abnormal accumulation of hematopoietic progenitors in the bone marrow (BM). The AML phenotype is induced and maintained by specific chromosomal translocations, such as t(8;21), t(15;17), t(6;9). These leukemia initiating events lead to an accelerated proliferation due to a differentiation block that prevents progenitors from reaching the post-proliferative stage of blast cells. This is supported by the aberrant stem cell capacity of poorly defined leukemic stem cells (LSC). The related AML associated fusion proteins (FPs) such as PML/RARα-t(15;17) (P/R), AML-1/ETO-t(8;21) or DEK/CAN-t(6;9) (D/C) recapitulate the leukemic phenotype in vitro and in vivo. Deregulated activation of the Wnt-signaling by FPs lead to aberrant self renewal of LSC in AML and is fundamental for maintenance of the LSC population. Wnt-signaling can be inhibited by non steroidal anti inflammatory drugs (NSAID, i.e. Sulindac, Indomethacin), mainly dual COX 1/2 inhibitors that in high concentrations, which are relevant for the Wnt-signaling inhibition also target the 5-Lipoxygenase (5-LO). Both COX1/2 and 5-LO are the key enzymes in the arachidonic acid metabolism. COX1/2 are the key enzymes for the synthesis of prostaglandines whereas 5-LO is responsible for the biosynthesis of leukotrienes, a group of pro-inflammatory lipid mediators. Reportedly the loss of 5-LO abolished the LSC of BCR/ABL induced CML-like disease. Therefore we targeted 5-LO by selective inhibitors (CJ 13,610 or Zileuton) and have recently shown that the inhibition of the 5-LO enzymatic activity interferes with the stem cell capacity of both PML/RARα and DEK/CAN positive LSC. In order to definitively show that the effects of the 5-LO inhibitors are related to the inhibition of 5-LO, we extended our studies on P/R- and D/C-positive LSC in a 5-LO−/−background. Here we show that loss of 5-LO expression not only failed to inhibit the stem cell activity of P/R or D/C expressing LSC, but significantly increased the colony number and the replating efficiency of D/C-positive LSC as compared to 5-LO+/+ controls. No significant differences were seen between short-term (ST) or long-term (LT) stem cell capacity of P/R or D/C expressing HSC in 5-LO−/− as compared to a 5-LO+/+ background. These data led us to the conclusion that the inhibition, but not the absence of 5-LO is important for the effect of 5-LO inhibitors on the Wnt-signaling. In order to prove the hypothesis that the inactive 5-LO is the inhibitor of the Wnt-signaling, we expressed 5-LO in U937 cells which are 5-LO-negative. We used U937 P/R9 cells, in which the inducible expression of PML/RAR strongly activates the Wnt-signaling as revealed by the Topflash/Fopflash transactivation system. The expression of 5-LO in absence of leukotriene stimulation reduced the Wnt-signaling activation by P/R to the levels seen in controls. These effects of 5-LO on Wnt-signaling can be explained by the direct interaction between 5-LO and β-catenin as revealed by co-immunoprecipitations in U937 cells. Taken together our data strongly suggest 1.) an important role of 5-LO in the regulation of the Wnt-signaling pathway and thereby for the maintenance of LSC and 2.) that the inhibitory effect of 5-LO inhibitors are not due to the interruption of the 5-LO mediated lipid signaling but to the presence of an inactivated 5-LO protein, which acquires novel functions that are disconnected from the lipid signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2964 Poster Board II-940 The t(9;22)-related translocation products are the BCR/ABL fusion proteins. Fusion of BCR to ABL leads to constitutive activation of ABL tyrosine kinase (ATK) activity. c-ABL is finely regulated by a variety of stimuli, whereas constitutively activated ABL induces aberrant proliferation and neoplastic transformation by constitutive activation of RAS, PI3 kinase, and JAK/STAT. Constitutively activated ATK is indispensable for the transformation potential of BCR/ABL. Inhibition of BCR/ABL kinase activity by selective ATP-competitors, such as Imatinib, Dasatinib or Nilotinib, is a valid concept for the causal therapy of Ph+ leukemia. Unfortunately, in advanced Ph+ leukemia, CML-blast crisis, and Ph+ ALL, these compounds select for resistant clones mostly due to the acquisition of point mutations in BCR/ABL that change the affinity for these ATP competitors. Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for aberrant ABL-kinase activation. We recently showed that targeting the CC-domain forces BCR/ABL into a monomeric conformation that abolishes its transformation potential by interfering with its kinase activity. This also increases the sensitivity of the leukemic cells to Imatinib and overcomes the Imatinib resistance of BCR/ABL harboring the Y253F and E255K mutations. Another mechanism to target BCR/ABL consists in the allosteric inhibition. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit BCR/ABL. The the “gatekeeper” mutation T315I confers pluri-resistance against ATP competitor, oligomerization as well as allosteric inhibitors Based on data showing that the response to GNF-2 varies with the oligomerization in ABL-chimeras we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. Therefore we combined GNF-2 with the competitive peptide helix-2, for which we have previously shown the capacity to target BCR/ABL and its Imatinib-resistant mutants. Here we demonstrate that i.) the helix-2 strongly increased the inhibitory effects of 2μM GNF-2 on the factor independent growth of BCR/ABL expressing Ba/F3 progenitors; ii.) both helix-2 and GNF-2 were able to decrease the transformation potential of BCR/ABL in Rat-l fibroblasts; iii.) the combination of helix-2 and GNF-2 completely abolished the colony formation of BCR/ABL-positive Rat-1 cells in semi-solid medium; iv.) in combination helix-2 and GNF-2 were able to abrogate factor independent growth of Ba/F3 cells expressing BCR/ABL-T315I, whereas no effect was seen by the single agents; v.) the helix-2/GNF-2 combination nearly completely abolished the transformation potential of BCR/ABL-T315I in Rat-1 cells; vi.) the inhibitory effects of helix-2/GNF-2 were due to an inhibition of the BCR/ABL dependent signaling as shown by a reduced activation of STAT5 and a block of substrate phosphorylation (Crkl). The fact that the helix-2 is able to overcome the resistance of BCR/ABL-T315I against GNF-2 strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1846 Chromosomal translocations such as t(15;17), t(8,21) or t(6;9) lead to the formation of chimeric genes encoding the PML/RAR, AML-1/ETO or DEK/CAN fusion proteins (FP). These FP are able to induce and to maintain acute myeloid leukemia (AML) by both blocking terminal differentiation of early hematopoietic progenitors and increasing the self renewal potential of the leukemic stem cells (LSC). LSCs are potential therapeutic targets and it is of great importance to elucidate which signaling pathways control their development and maintenance. Recently it has been shown that the presence of the 5-Lipoxygenase activity (5-LO) is indispensable for the induction and the maintenance of the BCR/ABL induced CML-like disease in mice. Its depletion or inhibition impairs the LSCs in the CML-like disease. 5-LO is the key enzyme in the biosynthesis pathway of leukotrienes, a group of proinflammatory lipid mediators derived from arachidonic acid. Furthermore we have shown that Sulindac sulfide, a dual Cycloxygenase/5 –LO inhibitor, was able, at 5-LO inhibitory concentrations, to interfere with the stem cell capacity of PML/RAR-positive LSC. It also overcame the differentiation block in PML/RAR-positive HSC. To disclose whether a “leukemic stem cell therapy” in AML is feasible if based on selectively targeting the 5-LO, we used two different selective 5-LO inhibitors, Zileuton and CJ-13,610, in a PML/RAR- and DEK/CAN-positive leukemia model. Zileuton, an anti-asthmatic drug, is a reversible inhibitor of 5-LO activity which leads to the inhibition of leukotrienes (LTB4, LTC4, LTD4, and LTE4) formation. CJ-13,610 is novel non redox, non iron chelating 5-LO inhibitor. As stem cell models we used Sca-1+/lin-murine HSC retrovirally transduced either with PML/RAR or DEK/CAN. Here we report that both Zileuton and CJ -13,610 at clinically feasible concentrations of 0.3 – 3μM interfered with the aberrant replating efficiency of PML/RAR and DEK/CAN expressing HSCs; ii.) inhibited the short-term stem cell (ST-HSC) capacity of PMR/RAR- and DEK/CAN-positive HSCs as assessed by colony forming unit-spleen day 12 assays in lethally irradiated recipient mice; and iii.) reduced the frequency of long-term HSC in a long term competitive repopulation stem cell assays. The effects of both compounds were not due related to the induction of apoptosis. Interestingly, on normal control HSC both Zileuton and CJ-13,610 exhibited a “paradox” effect by increasing ST-HSC as well as LT-HSC capacity. Our here presented data establish the inhibition of 5-LO by selective inhibitors as a feasible approach of molecular stem cell therapy in AML. Furthermore it strongly suggest an important role of leukotrienes for the maintenance of leukemic stem cells. The exact mechanisms by which the inhibition of 5-LO interferes with the LSC have still to be disclosed. Disclosures: Off Label Use: The use of anti-inflammatory drugs such as Zileuton and CJ-13610 as novel approach for stem cell treatment in AML is discussed.

Description: Abstract 1384 In acute myeloid leukemia (AML) chimeric genes derived from specific chromosomal translocations, such as t(15;17), t(8;21) or t(6;9) encode aberrant fusion proteins (FPs). These Fps, i.e. PML/RARα (PR), AML-1/ETO (AE) or DEK/CAN (DC), recapitulate the leukemic phenotype in the mouse. In about 50% af AMLs constitutive activation of STAT3 and/or 5 is observed and it is postulated that the aberrant stem cell capacity in leukemia is correlated to the activation of STATs. Furthermore it has been shown in models of the t(15;17)(PR)-positive AML that there is a relationship between STAT-activation and response to arsenic trioxide (ATO). t(6;9)(DC)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The mechanisms by which DC induces leukemia are nearly completely unknown. In order to investigate whether the STAT-activation plays a role for the pathogenesis of t(6;9)-positive AML we investigated the activation of STAT3 and 5 in models of DEK/CAN positive leukemia. As models of DC-induced AML we used either primary Sca1+/lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DC or as controls with PR. Samples of DC-positive pre-leukemia as well as primary leukemia and 2° leukemia from established DC-induced AML were used for the in vivo studies. Expression and activation of STAT3 and 5 was assessed by either intracellular FACS or Western blotting using antibodies against the phosphorylated (activated) and total STAT3 or 5. The effect of constitutively activated STAT3 (STAT3*) on the stem cell capacity was studied by the retroviral expression of STAT3* in mHSPC which then were subjected to serial replating and colony-forming unit spleen day 12 (CFU-S12) assays. ATO treatment was performed on sublethally irradiated recipients (8 mice/group), from day 5 after inoculation of DC-positive primary leukemic spleen cells for the induction of a 2° leukemia for 14 days. Leukemia development and response to ATO was assessed 30 days post-transplantation by spleen size and determination of the proportion of leukemic cells in the peripheral blood (PB), spleen and bone marrow (BM) in comparison to solvent treated controls. Here we show that i.) both DC and PR strongly activated both STAT3 and 5 in primary blasts from mouse leukemia as as well as in retrovirally transduced primary HSCPs; ii) the expression of constitutively activated STAT3 (STAT*) in HSC led to an increased replating efficiency in the presence of mIL-3, mIL-6 and SCF; iii.) STAT* increased CFU-S12 as a read out for short-term stem cell and early progenitor potential to a similar extent as PR. iv.) the pre-leukemic state of DC-positive AML was characterized by the only activation of STAT3 but not of STAT5; v.) STAT5 activation appeared only in the full blown leukemia; v.) exposure to ATO inhibited phosphorylation of STAT3 and STAT5 in vivo in the PB of the leukemic mice; vi.) the treatment with ATO significantly decreased spleen size and the proportion of leukemic cells in the BM and spleen as compared to solvent treated controls. In summary, our results strongly suggest a direct relationship between the expression of the leukemogenic oncogenes DC or PR and the activation of STATs, which seems to play an important role for the maintenance of the leukemic stem cell. Furthermore ATO seems to represent a novel therapeutic option for the t(6;9)-positive AML. Disclosures: No relevant conflicts of interest to declare.

Description: Targeting of BCR/ABL by ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib is a proven concept in Philadelphia chromosome positive (Ph+ ) leukemia. In the majority of cases the acquisition of resistance is related to point mutations in BCR/ABL, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Noteworthy, Ph+ leukemias, both CML and Ph+ ALL, never emerge at diagnosis with a BCR/ABL harboring a resistance mutation even if the clone with the mutation is already existing and detectable by very sensitive methods. This indicates that the mutations confer biological features to the clones unveiled by the treatment. One can hypothesize that the presence of mutations such as the T315I confers a growth disadvantage with respect to native BCR/ABL. We and others have previously shown that the resistance mutations may influence the biology of BCR/ABL regarding its transformation potential. The aim of the study was to determine whether the ‘‘gatekeeper’’ mutation T315I is able not only to induce biochemical modification of BCR/ABL responsible for the to resistance of patients against first and second generation AKI, but also to confer biological features to BCR/ABL influencing its leukemogenic potential. We recently showed that T315I is able to fully restore factor independent growth in Ba/F3 cells of loss of function mutants (LOFM) of BCR/ABL such as that lacking the Y177 (Y177F) or the N-terminal coiled coil oligomerization interface (ΔCC), which was accompanied by a transphosphorylation of endogenous BCR. Based on these findings we systematically investigated the influence of T315I on the biology of BCR/ABL and the role of BCR in this process. As models we used a syngeneic mouse model of BCR/ABL induced CML-like disease, factor dependent 32D and Ba/F3 cells, and Ph+ ALL patient derived long term culture (PD-LTCs). These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. Furthermore we took advantage of LOFM such as ΔCC-BCR/ABL and Y177F-BCR/ABL which we studied for their capacity to mediate either transformation potential in Rat-1 fibroblasts (contact inhibition and anchorage-independent growth) and/or factor independent growth in 32D cells in the presence of T315I. The role of the transphosphorylation of BCR was assessed by RNAi against BCR in 32D cells and the Rat-1 cells. Here we show that proliferation of 32D or Ba/F3 cells or the PD-LTCs expressing BCR/ABL-T315I was significantly slower than that of correspondent cells expressing native BCR/ABL. Also the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). On the other hand T315I was able to restore both transformation potential and factor independent growth of LOFM of BCR/ABL in Rat-1 and 32D cells, respectively. This was accompanied by a transphosphorylation of endogenous BCR at Y177, which led to an activation of Ras/Erk1/2 pathway. This effect of T315I on factor independent growth and transformation mediated by the LOFM and related activation of Ras/Erk1/2 was reverted by targeting the BCR with RNAi. Taken together these data suggest that T315I confer biological features to BCR/ABL which are unveiled only upon treatment or in the presence of LOFM. How these features may influence the destiny of the BCR/ABL-T315I clone in the patient and the role of Ras/Erk1/2 pathway in this process has to be further investigated. Disclosures No relevant conflicts of interest to declare.

Description: Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

Description: The chromosomal translocation t(4;11)(q21;q23) is the most frequent genetic aberration of the human MLL gene, resulting in high-risk acute lymphoblastic leukemia (ALL). To elucidate the leukemogenic potential of the fusion proteins MLL·AF4 and AF4·MLL, Lin−/Sca1+ purified cells (LSPCs) were retrovirally transduced with either both fusion genes or with MLL·AF4 or AF4·MLL alone. Recipients of AF4·MLL- or double-transduced LSPCs developed pro-B ALL, B/T biphenotypic acute leukemia, or mixed lineage leukemia. Transplantation of MLL·AF4- or mock-transduced LSPCs did not result in disease development during an observation period of 13 months. These findings indicate that the expression of the AF4·MLL fusion protein is capable of inducing acute lymphoblastic leukemia even in the absence of the MLL·AF4 fusion protein. In view of recent findings, these results may imply that t(4;11) leukemia is based on 2 oncoproteins, providing an explanation for the very early onset of disease in humans.