ALBERT

Description: Central nervous system (CNS) relapse accompanying prolonged administration of imatinib mesylate, an Abl-specific tyrosine kinase inhibitor, has recently become apparent as an impediment to the therapy of Philadelphia-chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib into the cerebrospinal fluid due to presence of P-glycoprotein (P-gp) at blood-brain barrier. To overcome imatinib-resistance mechanisms such as bcr-abl gene amplification, point mutations within ABL kinase domain, and activation of Lyn, we recently developed a specific dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25–55 times more potent than imatinib in vitro and at least 10 times more potent in vivo (Blood106: 3948–3954, 2005). The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. The intracellular accumulation of [14C]INNO-406 in P-gp overexpressing LLC-GA5-COL150 cells was much less than that in parental LLC-PK1 cells. The addition of 10 mM cyclosporin A (CsA) increased the intracellular accumulation of [14C]INNO-406 in both LLC-PK1 cells and LLC-GA5-COL150 cells. The peak concentration of INNO-406 in the brain when 30 mg/kg INNO-406 was administered p.o. was 50 ng/ g (87 nM), representing only 10% of plasma drug level. These findings suggested that INNO-406 is also a substrate of P-gp, as is imatinib. However, the residual concentration of INNO-406 in the CNS was enough to inhibit the growth of Ph+ leukemic cells according to the in vitro data. To increase the concentration of INNO-406 in CNS, we next examined the combined effects of CsA. In the brain, the concentration of INNO-406 was doubled following prior administration of 50 mg/kg CsA. Since pharmacokinetic studies suggested the possible effects of INNO-406 against CNS Ph+ leukemia, we investigated in vivo anti-CNS Ph+ leukemia effects of INNO-406 alone and combination of INNO-406 and CsA using immunodeficient mice (nude or NOD/SCID) which received Ph+ leukemic cells into the cerebral ventricle. INNO-406 alone inhibited growth of leukemic cells harboring either wild type or mutated BCR-ABL such as E255K and M351T in CNS. Furthermore, CsA significantly enhanced anti-CNS Ph+ leukemia effects of INNO-406 in vivo not only against cells harboring wild type BCR-ABL but also against cells harboring BCR-ABL/M351T (Figure). In conclusion, INNO-406 was found to inhibit Ph+ leukemic cell growth in CNS in spite of efflux of the compound by P-gp, and CsA augmented the anti-CNS Ph+ leukemia effects of INNO-406. Phase I clinical study on INNO-406 was initiated in the U.S.A. in July 2006. The efficacy and safety of INNO-406 in the treatment of leukemias is expected to be verified by early-phase clinical trials. Figure Figure

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: Central nervous system (CNS) is one of the major cites for extramedullary relapse of Ph+ leukemias, which have been treated with imatinib mesylate (IM). The reason for this is that IM is a substrate for P-glycoprotein (P-gp) at the blood brain barrier and effluxed by it. We have already shown in the last annual meeting that INNO-406 had much stronger anti-tumor effects against the murine CNS leukemia model compared with IM, and INNO-406 is also effluxed from the murine CNS by P-gp. In this study, we investigated the combination effect of INNO-406 and P-gp inhibitors, verapamil or cyclosporin-A (CsA). First, we examined the growth-suppressive effect of INNO-406 and the combination with the P-gp inhibitors against the BCR-ABL positive leukemic cell line, K562 and the P-gp-overexpressing K562, K562/D1-9 cell line. K562/D1-9 showed 10 times higher resistant to both IM and INNO-406 compared with K562. Furthermore, both verapamil and CsA synergistically augmented the effect of INNO-406. Next, we investigated the pharmacokinetics of INNO-406 when orally administrated with CsA to mice. Mice were administrated p.o. with 50mg/kg of CsA 2 hours before INNO-406. We found that the concentration of INNO-406 in the CNS elevated by 2 times when combined with CsA, while the plasma concentration was decreased to two thirds of that when singly administrated with INNO-406. It was suggested that the decreased plasma concentration of INNO-406 seen here resulted from the increased uptake into the CNS by CsA inhibiting P-gp at the blood brain barrier. These changes of the drug distribution to the murine tissues may alter the anti-leukemic effect of INNO-406, thus we planned to investigate the combination effect of INNO-406 and CsA in the murine models of both CNS and systemic leukemia. We found that CsA significantly augmented the anti-tumor effect of INNO-406 in the CNS leukemia model. Moreover, in spite of the decreased plasma concentration of INNO-406, the combination with CsA also prolonged the survival phase of the mice in the systemic leukemia model, more significantly than single treatment of INNO-406 (Figure). This may be explained by which CsA increased the intracellular uptake of INNO-406, resulted from the direct inhibition of drug efflux via P-gp expressed in the leukemic cells. Phase I clinical study on INNO-406 is now underway in MD Anderson Cancer Center and in Frankfurt University. From the results of this study, we expected the effective application of INNO-406 in combination with P-gp inhibitor to the patients suffering from refractory Ph+ leukemia as well as CNS relapse. Figure Figure

Description: Acute promyelocytic leukemia (APL) is distinguished from other AMLs by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17) which leads to the expression of the PML/RAR fusion protein. PML/RAR is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA). Patients suffering from APL undergo complete hematologic but not molecular remission upon treatment with t-RA. Virtually all patients treated with t-RA-monotherapy had a rapid relapse within few months. But in the combination with an anthracycline, such as doxorubicin or idarubicin, t-RA improved the long term outcome of APL-patients dramatically. Nothing is known about why t-RA-monotherapy is unable to eradicate completely the leukemic population and how it increases the response to chemotherapy. In vitro, the exposure of early hemopoietic stem cells (HSCs) to t-RA does not induce differentiation but selects immature progenitors. Moreover, mice lacking the t-RA-specific receptor RARalpha do not exhibit an impairment of granulopoiesis or hemopoiesis. The indication, that t-RA may be involved in the hemopoietic differentiation, is given by the HL-60 cell line which undergoes granulocytic differentiation at the pharmacological dosages (10−6M) of t-RA. Furthermore vitamin A-deficient mice or mice treated with a antagonist of t-RA accumulate more immature granulocytes in the bone marrow. PML/RAR mediates the response of APL blasts to t-RA, but it is completely unclear, which effect t-RA exerts on the PML/RAR-positive leukemic stem cells which maintains the blast population and represents the source of relapse. Therefore we investigated the effect of t-RA on a cell population with stem cell capacity expressing PML/RAR isolated from the APL cell line NB4 as well as from CD34+/CD38- KG-1 cells transfected with PML/RAR. Here we report that i) the NB4 cells engrafted in NOD/SCID mice indicating the presence of a subpopulation with stem cell capacity in NB4 cells; ii) NB4 had a Hoechst 3342 excluding side population (SP) representing about 1% of the whole cell population; iii) t-RA reduced but did not deplete the side population in NB4 cells; iv) the expression of PML/RAR increased CD34+/CD38- population in KG-1 cells from 75% to over 95%; v) t-RA reduced the CD34+/CD38- population from 75% to 3,5% in mock transfected KG-1 confirming its capacity to induce differentiation, whereas in PML/RAR-positive KG-1 cells it led only to a reduction from 98% to a 25%, which still maintain the capacity to engraft in NOD-SCID mice; vi) also the expression of other fusion proteins, such as AML-1/ETO or PLZF/RAR, associated with t-RA-resistant AML-subtypes, increased the percentage of CD34+/CD38- KG-1 cells over 90%, which was reduced by t-RA only to 35% and 19%, respectively. Taken together these data suggest that a subset of early HSC expressing PML/RAR exhibit the same t-RA-resistant phenotype as HSC expressing fusion proteins associated with AML-subtypes which, in contrast to APL, do not respond to t-RA. These data may give an explanation, why APL-patients do not achieve complete molecular remission upon t-RA monotherapy and undergo early relapse.

Description: Background Targeting BCR/ABL by ABL-directed kinase inhibitors (AKIs) induces long lasting remissions in patients with chronic myeloid leukemia (CML), and short remissions in Ph+ acute lymphatic leukemia (ALL). Notably in advanced Ph+ leukemia resistance attributable to either kinase domain mutations in BCR/ABL or non mutational mechanisms remains the major clinical challenge. With the only exception of Ponatinib, a multitargeted kinase inhibitor, all actually approved AKIs are unable to inhibit the „gatekeeper“ mutation T315I. Ponatinib is unable to overcome non mutational resistance in advanced leukemia and has an unfavorable spectrum of undesired side effects, most likely due to the broad spektrum of kinase inhibition. Thus there is the urgent need for further and more selective therapeutical options in treating therapy-resistant advanced Ph+ leukemia. PF114 is an ATP competitor, which was developed focusing on: i.) targeting all known resistance mutations in BCR/ABL but mainly the T315I; ii.) a higher selectivity as compared to Ponatinib in order to reduce undesired side effects; iii.) the ability to overcome also non mutational resistance in advanced Ph+ leukemia; iv.) an activity not only in chronic phase CML but also advanced Ph+ leukemia, Ph+ ALL or blast crisis CML (BC-CML). Methods An toxicity profile of PF-114 as well as its kinase selectivity was investigated. The preclinical evaluation of PF-114 was performed in direct comparison to Ponatinib on golden standard preclinical models of CML and advanced Ph+ leukemia and primary patient-derived long term cultures (PD-LTC) of Ph+ ALL patients. The effects on mutational resistance was investigated i.) on the factor dependence of Ba/F3 cells expressing BCR/ABL or its clinically most relevant resistance mutants (Y253F, E255K, T315I, F317L); ii.) on a PD-LTC of a Ph+ ALL patient harboring the T315I. As models for non mutational resistance we used PD-LTCs from Ph+ ALL patients with different levels of non mutational drug resistance and the SupB15RT, a Ph+ ALL cell line rendered resistant by the exposure to increasing doses of Imatinib and cross-resistant against all approved AKIs. The effects of PF114 in vivo were investigated on the transduction/transplantation model of BCR/ABL- and BCR/ABL-T315I-induced CML-like disease, secondary BCR/ABL-induced murine ALLs as well as on xenografts of PD-LTCs in NSG mice and K562 cells in nude mice. Results PF-114 is an orally available AKI, which is more selective than Dasatinib or Ponatinib (number of kinases inhibited at 100 nM of a drug: Nilotinib - 19; PF-114 - 27; Dasatinib - 48; Ponatinib - 80). It was classified as a class 4 - low-toxic - substance (Hodge/Sterner classification). It efficiently inhibited all tested BCR/ABL mutants in cellular and biochemical assays at dosages of 10-100nM and like Ponatinib it suppressed the up-coming of new resistance mutations in a mutation assay in Ba/F3 cells. It also suppressed growth of Ph+ PD-LTC with non mutational resistance as well as the BCR/ABL-T315I-positve PD-LTC in this dosage range. In all models the effect was independent of the presence of either the p210 or the p185 form of BCR/ABL. No effect of PF114 was seen in PD-LTCs of Ph- ALL. Noteworthy PF-114 (50 mg/Kg) prolonged significantly the survival of mice with both BCR/ABL- and BCR/ABL-T315I-driven CML-like disease as compared to Ponatinib (25 mg/Kg). In all in vivo models of advanced leukemia, PF-114 (50 mg/Kg) significantly inhibited leukemia to a similar extent as Ponatinib (25 mg/Kg). Like Ponatinib, PF-114 was unable to overcome the non mutational resistance in SupB15RT. Conclusions Our work supports clinical evaluation of PF114 as a pan BCR/ABL inhibitor for treatment not only for chronic phase CML, but also for advanced and resistant Ph+ leukemia such as Ph+ ALL or BC-CML. Disclosures: Mian: Fusion Pharma: Research Funding. Zeifman:Fusion Pharma: Employment. Titov:Fusion Pharma: Employment. Stroylov:Fusion Pharma: Employment. Stroganov:Fusion Pharma: Employment. Novikov:Fusion Pharma: Employment. Chilov:Fusion Pharma: Employment. Ruthardt:Fusion Pharma: Research Funding.

Description: Abstract 598 The t(9;22) translocation (Ph+) leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. Depending on the minor and major breakpoint (m-BCR or M-BCR, respectively) on chromosome 22, either the p185-BCR/ABL (p185) or the p210-BCR/ABL (p210) fusion protein is expressed. In contrast to p210, p185 lacks the putatively oncogenic dbl- and pleckstrin homology domains. In the majority of the cases Ph+ ALL patients harbor the p185, but with increasing age the number Ph+ ALL patients with the p210 increases up to an 40%. Only few functional and biological differences between p185 and p210 are known. Both exhibit a constitutively activated kinase activity responsible for the induction of the leukemic phenotype in contrast to their physiological counterpart, c-ABL, whose kinase activity is finely regulated. One reason is that BCR/ABL “escapes” auto-inhibition mechanisms of c-ABL, such as the allosteric inhibition by “capping”. “Capping” is the process by which myristoylation of the N-terminal cap region of c-ABL leads to an auto-inhibited conformation through its binding to the hydrophobic myristoyl binding pocket (MBP) in the kinase domain. The cap region of c-ABL is encoded by the exon I, which is replaced by the N-terminus of BCR in the fusion protein. Myristate “mimicks” and MBP binders, such as GNF-2, aim to restore the allosteric inhibition through the binding to the MBP in BCR/ABL. The aim of our study was to further develop allosteric inhibition in Ph+ ALL. Therefore we investigated the anti-proliferative activity of GNF-2 in different Ph+ leukemia models such as patient derived Ph+ lymphatic cell lines, long term cultures of Ph+ ALL patient derived bone marrow cells (LTC) and factor dependent lymphatic Ba/F3 cells retrovirally transduced with either p185 or p210. Here we report that i.) the IC50 of GNF-2 for p210-positive ALL cells were much lower (cell lines =125nM; LTC from two different patients 100–300nM) than that for p185-positive ALL cells (cell lines=500 nM; LTC from two different patients 500–1000nM); ii.) factor independent growth of Ba/F3 cells expressing p185 was inhibited with an IC50 of 750nM whereas that of Ba/F3 cells expressing p210 was abolished already at 125nM; iii.) 1000nM GNF-2 was able to abolish colony formation in soft agar of p210-positive but not of p185-positive LTC; iv.) increasing concentrations of GNF-2 abolished in p210-positive ALL cells the BCR/ABL kinase activity, whereas in p185-positive cells it only reduced it as shown by the effects of GNF-2 on the autophosphorylation (Y245) and substrate phosphorylation (Crkl) activity as well as on down stream signaling (Stat5) of BCR/ABL. Our here presented data provide first evidence of a differential response of p185- and p210-positive Ph+ ALL cells to the allosteric inhibition by GNF-2, as a expression of functional and biological differences between the two fusion proteins within the same disease entity. It remains to clarify not only the mechanisms of this differential response, such as three dimensional conformation, different kinase activity and related signal transdcution, but also its prognostic significance for patients with p210-positive Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.