Neue Ansätze des molekularen Targetings bei der Philadelphia-Chromosom-positiven Leukämie

In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, reduces its kinase activity and increases the sensitivity for Imatinib. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; iii.) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. 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 unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. 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.In der Philadelphia Chromosome (Ph) positiven ALL und CML hat die Fusion von BCR und ABL die Bildung eines BCR/ABL Fusionsprotein zur Folge. Dieses Fusionsprotein ist für den leukämischen Phänotypen aufgrund der konstitutiven Aktivierung vieler Signalwege unterhalb/Downstream der veränderten BCR/ABL fusionierten Tyrosinkinase.Eine zielgerichtete Inhibierung von BCR/ABL mittels ABL-Kinase-Inhibitoren induziert Apoptose in BCR/ABL transformierten Zellen und hat eine komplette Remission in Ph+ Leukämie Patienten zur Folge. Eine große Anzahl an Patienten mit fortgeschrittener Ph+ Leukämie erleiden einen Rückfall und entwickeln Resistenzen. Die Mutation der Kinasedomäne verhindert die Bindung von Inhibitoren und stellt somit den häufigsten Mechanimus von erworbenen Resistenzen in Patienten mit Ph+ Leukämie. Die Tetramerizierung von BCR/ABL mittels der N-Terminalen coiled-coil region (CC) von BCR ist notwendig für die Aktivierung der ABl-Kinase. Das Targeting der CC-Domäne zwingt BCR/ABL in eine monomere Konformation, was zu einer Reduzierung der Kinasaktivität und einer erhöhten Imatinib-Sensitivität führt. Wir können zeigen, daß i) das Angreifen der Tetramerisierung mittels einem Peptide, welches die Helix2 der CC-Domäne repräsentiert, reduziert die Autophosphorylierung sowohl von WT BCR/ABL, als auch seinen Mutanten. ii) Die Helix-2 inhibiert unabhängig von vorkommenden Mutationen das transformierende Potential von BCR/ABL. iii) Aufgrund der Effekte auf das transformierende Potential und dem Faktor unabhängigen Wachstum von BCR/ABL, mit Ausnahme der T315I-Mutation, konnte eine effektive Kooperation zwischen Helix-2 und Imatinib gezeigt werden. Die gezielte Hemmung von BCR/ABL durch Inhibitoren in Form kleiner Moleküle macht das Potential zur Transformation von BCR/ABL rückgängig. Wir haben eindeutig bewiesen, dass das Zielen auf die, durch die N-terminale coiled-coil Domäne (CC) vermittelte, Tetramerisierung von BCR/ABL mithilfe kompetitiver Peptide, welche die Helix-2 von CC repräsentieren, ein wirkungsvoller therapeutischer Ansatz zur Behandlung der Ph+Leukämie ist. Um die kompetitiven Peptide zum Angriff auf BCR/ABL weiter zu entwickeln, erzeugten wir eine Membran permeables Helix-2 Peptid (MPH-2), indem wir die Helix-2 Peptide mit einem Peptid-Transduktions-Tag fusionierten. In dieser Studie berichten wir, dass MPH-2: (i) mit BCR/ABL in vivo interagierte; (ii) effizient die Autophosphorylierung von BCR/ABL hemmte; (iii) Wachstum und Viabilität von Ph+ leukämischen Zellen unterdrückte; und (iv) in einem in vivo Maus-Modell effizient in mononukleäre Zellen (MNC) transduziert wurde. Die T315I Mutation von BCR/ABL weist eine Resistenz gegen alle momentan sich in medizinischen Studien befindlichen ABL-kinase Inhibitoren und kompetitiven Peptiden auf. Es scheint nicht nur die Bindungsaffinität der Kinase Inhibitoren zu vermindern sondern erzeugt zusätzliche Eigenschaften, die das leukämogene Potential von BCR/ABL verstärken. Um die Rolle der T315I Mutation auf dessen fehlender Inhibition der für das leukämogene Potential wichtigen Oligomerisierung von BCR/ABL näher zu bestimmen, untersuchten wir seinen Einfluß auf zusätzliche “loss-of-function” Mutanten von BCR/ABL in bezug auf deren Fähigkeit eine Faktor-Unabhängigkeit zu erzeugen. Entsprechend erzeugten wir BCR/ABL Mutanten, denen notwendige funktionale Domänen bezüglich des onkogenen Potenzials fehlen. Im BCR-Anteil deletierten wir die N-terminale coiled coil (CC) Domäne, die Tyrosine Phosphorylierungs Stelle Y177 und die Serine/Threonine Kinase Domäne (ST) sowie im ABL_Anteil die inhibitorische SH3 (delta SH3-ABL) Domäne. Aus den vorliegenden Arbeiten ergaben sich folgende Ergebnisse i.) T315I stellt die fehlende Faktor Unabhängigkeit von hämatopoetischen Zellen mit Oligomerizations defizienten p185BCR/ABL Mutanten wieder her; ii.) Die Resistenz von p185-T315I gegen die Inhibition der Oligomerisation ist abhängig von der Phosphorylierung am Y177; iii.) Die Autophosphorylierung am Y177 wird durch die Inhibition der Oligomerisierung nicht beeinträchtigt, jedoch die Phosphorylierung am Y177 von endogenem BCR wird durch die T315I Mutation verstärkt; iv.) Die Effekte von T315I sind mit einer intakten ABL_Kinase Aktivität assoziiert; v.) Das Vorhandensein der T315I Mutation ist assoziiert mit einer verstärkten ABL_kinase Aktivität, welches sich auch in Mutanten zeigt, die die Y177 Phosphorylierung von endogenem BCR nicht induzieren; vi.) Es gibt keinen direkten Zusammenhang zwischen der ABL-Kinase Aktivität und der durch T315I induzierten Faktorunabhängigkeit. Im Gegensatz zu der ABL-T315I Mutante, die nur in der Anwesenheit der SH3 Domäne nicht in der Lage war eine Phosphorylierung am Y177 von endogenem BCR zu induzieren. Im Gegensatz zu seinem physiologischen Pendant c-Abl, ist die BCR/ABL Kinase, die den leukämischen Phänotyp induziert, konstitutiv aktiviert. Der N-Terminus von c-ABL (Cap region) wirkt an der Regulation seiner Kinasefunktion mit. Er ist myristoyliert und in einem Prozess, genannt „capping“ bindet der Myristinsäurerest an eine hydrophobe Tasche in der Kinasedomäne, bekannt als Myristinsäurebindungstasche. Daraus resultiert eine autoinhibitorische Konformation. Da die Cap-region durch den N-terminus von BCR/ABL ersetzt wurde, entgeht BCR/ABL dieser Autoinhibition. Allosterische Inhibition durch Myristeinimitatoren, wie GNF-2, sind in der Lage nicht mutiertes BCR/ABL zu inhibieren, nicht jedoch BCR/ABL, das die „Gatekeeper“-Mutation T315I beherbergt. Wir untersuchen hier die Möglichkeit die Effizienz der allosterischen Inhibtion durch Blockierung der BCR/ABL-Oligomerisierung zu erhöhen. Wir demonstrieren, dass die Inhibtion der Oligomerisierung nicht nur in der Lage war die Effizienz von GNF-2 auf das nicht mutierte BCR/ABL zu erhöhen, sondern auch die Resistenz von BCR/ABL-T315I zu überwinden hin zur allosterischen Hemmung. Diese Ergebnisse lassen stark annehmen, dass das Ansprechen der allosterischen Hemmung durch GNF-2 in umgekehrtem Verhältnis zum Grad der Oligomerisierung von BCR/ABL steht. Zusammengenommen deuten diese Daten auf eine Inhibierung der Tetramerizierung hin, welsche die BCR/ABL vermittelte Transformation hemmt. Diese führt zu einer Überwindung der Imatinib Resistenz. Diese Studie bietet erste Evidenz, dass ein effizientes Peptid-Transduktions-System die Anwendung von kompetitiven Peptiden, um auf die Oligomerisierungs-Schnittstelle von BCR/ABL zu zielen, in vivo unterstützt. Des weitern zeigen die Daten, daß T315I zusätzliche leukämische Aktivität von BCR/ABL induziert, welches eine mögliche Erklärung für klinische Prognose von Patienten mit BCR/ABL-T315I positiven Blasten darstellt. Zusammenfassend lässt sich sagen, dass unsere Beobachtungen einen neuen Ansatz für molekulare Angriffspunkte für BCR/ABL und seine resistenten Mutanten etablieren, bestehend aus der Kombination von Oligomerisierungs- und allosterischen Inhibitoren

p185(BCR/ABL) has a lower sensitivity than p210(BCR/ABL) to the allosteric inhibitor GNF-2 in Philadelphia chromosome-positive acute lymphatic leukemia

Background: The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins are encoded as a result of the translocation, depending on whether a "minor" or "major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL) exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia. Design and methods: We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their resistance mutants. Results: The inhibitory effects of GNF-2 differed constantly between p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring resistance mutations. Conclusions: Our data provide the first evidence of a differential response of p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia

Targeting the oligomerization of BCR/ABL by membrane permeable competitive peptides inhibits the proliferation of Philadelphia Chromosome positive leukemic cells

The BCR/ABL fusion protein is the hallmark of Philadelphia Chromosome positive (Ph+) leukemia. The constitutive activation of the ABL-kinase in BCR/ABL cells induces the leukemic phenotype. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. Recently, we definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable helix-2 peptide (MPH-2) by fusing the helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. This study provides the first evidence that an efficient peptide transduction system facilitates the employment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo

Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors

Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism

ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells

Nano Titania Applications in Cancer Theranostics

Titanium is one of the most abundantly utilized nanomaterials for human consumption. Biomedical applications of nano titania include sunscreens, drug delivery, prosthetic implants, bioimaging probes, and antimicrobial and antirheumatic agents for various treatment of diseases, including autoimmune disease, neurogenerative diseases, cardiovascular, musculoskeletal, and cancer. Its applications as a drug delivery vehicle and photosensitizer in cancer therapy and diagnosis are highly appreciated, especially for skin and natural cavities applications. The reactive oxygen species (i.e., H2O2, OH., OH2, 1O2, etc.) generation properties of nano titania after activation with light or ultrasound make it ideal for apoptosis induction in neoplastic cells. In addition, the singlet oxygen (1O2) generating properties make it suitable for bioimaging deep-seated and superficial tumors after activation. Nano titania is highly biocompatible with negligible adverse effects. In this chapter, we will focus on the anticancer effects of nano titania on various types of cancers by employing it as a drug delivery vehicle and sensitizer for external source-activated modalities viz. photodynamic and sonodynamic therapy

Activation of AKT/mTOR pathway in Ph+ acute lymphoblastic leukemia (ALL) leads to non-mutational resistance

Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL.Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines.Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM.Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance