scholarly journals PSEN1-selective gamma-secretase inhibition in combination with kinase or XPO-1 inhibitors effectively targets T cell acute lymphoblastic leukemia

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Inge Govaerts ◽  
Cristina Prieto ◽  
Charlien Vandersmissen ◽  
Olga Gielen ◽  
Kris Jacobs ◽  
...  
Keyword(s):  
Cell Lines ◽  
Combination Treatment ◽  
Survival Benefit ◽  
Lymphoblastic Leukemia ◽  
Treatment Strategies ◽  
Gamma Secretase ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Targeted Inhibitors ◽  
Pdx Models

Abstract Background T cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype that comprises 10–15% of childhood and 20–25% of adult ALL cases. Over 70% of T-ALL patients harbor activating mutations in the NOTCH1 signaling pathway and are predicted to be sensitive to gamma-secretase inhibitors. We have recently demonstrated that selective inhibition of PSEN1-containing gamma-secretase complexes can overcome the dose-limiting toxicity associated with broad gamma-secretase inhibitors. In this study, we developed combination treatment strategies with the PSEN1-selective gamma-secretase inhibitor MRK-560 and other targeted agents (kinase inhibitors ruxolitinib and imatinib; XPO-1 inhibitor KPT-8602/eltanexor) for the treatment of T-ALL. Methods We treated T-ALL cell lines in vitro and T-ALL patient-derived xenograft (PDX) models in vivo with MRK-560 alone or in combination with other targeted inhibitors (ruxolitinib, imatinib or KPT-8602/eltanexor). We determined effects on proliferation of the cell lines and leukemia development and survival in the PDX models. Results All NOTCH1-signaling-dependent T-ALL cell lines were sensitive to MRK-560 and its combination with ruxolitinib or imatinib in JAK1- or ABL1-dependent cell lines synergistically inhibited leukemia proliferation. We also observed strong synergy between MRK-560 and KPT-8602 (eltanexor) in all NOTCH1-dependent T-ALL cell lines. Such synergy was also observed in vivo in a variety of T-ALL PDX models with NOTCH1 or FBXW7 mutations. Combination treatment significantly reduced leukemic infiltration in vivo and resulted in a survival benefit when compared to single treatment groups. We did not observe weight loss or goblet cell hyperplasia in single drug or combination treated mice when compared to control. Conclusions These data demonstrate that the antileukemic effect of PSEN1-selective gamma-secretase inhibition can be synergistically enhanced by the addition of other targeted inhibitors. The combination of MRK-560 with KPT-8602 is a highly effective treatment combination, which circumvents the need for the identification of additional mutations and provides a clear survival benefit in vivo. These promising preclinical data warrant further development of combination treatment strategies for T-ALL based on PSEN1-selective gamma-secretase inhibition.

scholarly journals Inducible transgene expression in PDX models in vivo identifies KLF4 as therapeutic target for B-ALL

2019 ◽  
Author(s):  
Wen-Hsin Liu ◽  
Paulina Mrozek-Gorska ◽  
Tobias Herold ◽  
Larissa Schwarzkopf ◽  
Dagmar Pich ◽  
...  
Keyword(s):  
Residual Disease ◽  
Therapeutic Option ◽  
Lymphoblastic Leukemia ◽  
Clinical Model ◽  
Novel Technique ◽  
Pdx Model ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Disease Stages ◽  
Pdx Models

Clinic-close methods are not available that prioritize and validate potential therapeutic targets in individual tumors from the vast bulk of descriptive expression data. We developed a novel technique to express transgenes in established patient-derived xenograft (PDX) models in vivo to fill this gap. With this technique at hand, we analyzed the role of transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive pre-clinical in vivo trials, we found that re-expression of wild type KLF4 reduced leukemia load in PDX models of B-ALL, with strongest effects after conventional chemotherapy at minimal residual disease (MRD). A non-functional KLF4 mutant had no effect in this model. Re-expressing KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. Of major translational relevance, Azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced Azacitidine-induced cell death, suggesting that Azacitidine can regulate KLF4 re-expression. These results support applying Azacitidine in patients with B-ALL to regulated KLF4 as a therapeutic option. Taken together, our novel technique allows studying the function of dysregulated genes in a highly clinic-related, translational context and testing clinically applicable drugs in a relevant pre-clinical model.

Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4630-4630
Author(s):  
Samuel D Gusscott ◽  
Florian Kuchenbauer ◽  
Andrew P Weng
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Protein Levels ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Efficacy of JAK1/2 and BCL2 inhibition on human T cell acute lymphoblastic leukemia in vitro and in vivo

2019 ◽  
Author(s):  
Kirsti L. Walker ◽  
Sabrina A. Kabakov ◽  
Fen Zhu ◽  
Myriam N. Bouchlaka ◽  
Sydney L Olson ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Human T Cell ◽  
Liquid Chromatography Tandem Mass ◽  
Cell Acute Lymphoblastic Leukemia

AbstractRelapsed/refractory T cell acute lymphoblastic leukemia (T-ALL) is difficult to salvage especially in heavily pretreated patients, thus novel targeted agents are sorely needed. Hyperactivated JAK/STAT and BCL2 overexpression promote increased T-ALL proliferation and survival, and targeting these pathways with ruxolitinib and venetoclax may provide an alternative approach to achieve clinical remissions. Ruxolitinib and venetoclax show a dose-dependent effect individually, but combination treatment synergistically reduces survival and proliferation of Jurkat and Loucy cells in vitro. Using a xenograft CXCR4+ Jurkat model, the combination treatment fails to improve survival, with death from hind limb paralysis. Despite on-target inhibition by the drugs, histopathology demonstrates increased leukemic infiltration into the central nervous system (CNS), which expresses CXCL12, as compared to liver or bone marrow. Liquid chromatography-tandem mass spectroscopy shows that neither ruxolitinib nor venetoclax can effectively cross the blood-brain barrier, limiting efficacy against CNS T-ALL. Deletion of CXCR4 on Jurkat cells by CRISPR/Cas9 results in prolonged survival and a reduction in overall and neurologic clinical scores. While combination therapy with ruxolitinib and venetoclax shows promise for treating T-ALL, additional inhibition of the CXCR4-CXCL12 axis will be needed to eliminate both systemic and CNS T-ALL burden and maximize the possibility of complete remission.

Growth Factor Independent-1 (Gfi1) Is Critically Required for T-Cell Acute Lymphoblastic Leukemia (T-ALL) Tumor Initiation and Maintenance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3156-3156
Author(s):  
James D. Phelan ◽  
Cyrus Khandanpour ◽  
Shane Horman ◽  
Marie-Claude Gaudreau ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Tumor Regression ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Apoptotic Genes

Abstract Abstract 3156 T cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers associated with mutations in NOTCH1. The Growth factor independent-1 (Gfi1) transcriptional repressor gene was originally discovered as a common target of Moloney murine leukemia virus (MMLV) proviral insertion in murine T-ALL. In fact, the Gfi1 locus is the most frequently activated gene in MMLV-induced T cell leukemia. Therefore, we investigated whether the most commonly activated gene in MMLV-induced murine T-ALL, Gfi1, could collaborate with the most commonly activated gene in human T-ALL, NOTCH1. Here, we show that GFI1 expression is associated with Notch signaling in human T-ALL (p'0.0003). Functionally, Gfi1 collaborates with Notch-induced murine T-ALL by accelerating an already rapid disease model (p=0.03) without altering the lymphoblastic nature of the disease. Furthermore, inducible deletion of Gfi1 is counter-selected in both Notch-driven retroviral and transgenic mouse models of T-ALL; whereas, constitutive absence of Gfi1 completely prevents transgenic Notch-induced T-ALL (p≤0.04). However, T-ALL tumors can form in Gfi1-/- animals using either ENU-mutagenesis or MMLV-infection, yet tumor formation is delayed (p≤0.02, p≤0.03 respectively). This suggests that Gfi1 deletion does not prevent the formation of the T-ALL initiating cell and that Gfi1 might be absolutely required for Notch-induced T-ALL. Most striking is that Gfi1 is required for T-ALL maintenance in vitro and in vivo. Using three separate Tal1-initiated murine T-ALL cell lines, the overexpression of the Gfi1 dominant-negative, Gfi1N382S, was quickly and completely counter-selected. As Gfi1 has previously been found to regulate pro-apoptotic genes in T cells, we attempted to rescue the above loss of function phenotype by overexpressing the anti-apoptotic factor Bcl2. Notably, counter-selection of Gfi1N382 is not observed or is significantly delayed in all three cell lines. In vivo, inducible deletion of Gfi1 leads to both mutagen- or Notch-induced tumor regression as measured by ultrasound. In fact, levels of Gfi1 expression directly correlate to tumor regression and disease free survival of T-ALL. Finally, targeting Gfi1 enhances the efficacy of radiation therapy and bone marrow transplantation. Deletion of Gfi1 sensitizes T-ALL tumors and T cells to p53-dependent apoptosis after exposure to DNA-damaging agents such as radiation, Etoposide or Daunorubicin by de-repression of the pro-apoptotic Gfi1 target gene Bax. These data extend the role of Gfi1 to human T-ALL and suggest that T-ALL is dependent upon Gfi1 to repress pro-apoptotic genes for tumor survival, ultimately highlighting a new therapeutic target in the fight against lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Wen-Hsin Liu ◽  
Paulina Mrozek-Gorska ◽  
Anna-Katharina Wirth ◽  
Tobias Herold ◽  
Larissa Schwarzkopf ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Residual Disease ◽  
Therapeutic Option ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Targets ◽  
Pdx Model ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Disease Stages ◽  
Pdx Models

Abstract Background Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. Methods We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. Results With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. Conclusion Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.

Direct Inhibition of the Notch Transactivation Complex with Synthetic Constrained Peptides in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2819-2819 ◽  
Author(s):  
Raymond Moellering ◽  
Melanie Cornejo ◽  
Jennifer Rocknik ◽  
Michael Hancock ◽  
Christina DelBianco ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Stem Cell Differentiation ◽  
Gamma Secretase ◽  
Clinical Resistance ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

scholarly journals Targeting a Specific Glycosylated Epitope of CD43 with a New Humanized Monoclonal Antibody for the Treatment of Pediatric and Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1418-1418
Author(s):  
Cirino Botta ◽  
Giuseppe Gaipa ◽  
Maria Eugenia Gallo Cantafio ◽  
Chiara Buracchi ◽  
Maria Anna Siciliano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Target Cells ◽  
In Vivo Models ◽  
Normal Tissues ◽  
Healthy Donors ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 20% of pediatric and adult ALL cases. Despite the use of intensive chemotherapy protocols, 25% of children and 50% of adult patients fail to respond or relapse. The 3-years prognosis for these patients is poor and novel treatment options are needed. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) is among the most investigated immune-therapeutic strategies. Accordingly, we developed a new humanized mAb (hUMG1), directed against a heavy glycosylated epitope of CD43 which presents a high reactivity against T-ALL cells. Here we investigated the pre-clinical therapeutic activity and the mechanisms of action of hUMG1 in experimental models of T-ALL. Methods: The expression of hUMG1 target was assessed by flow cytometry on tumor cell lines and primary samples from either T-ALL patients (n=48) or healthy donors (n =6). Humanized mAbs were generated by combining the variable domains of the murine antibody to the corresponding human IgG1 constant domains. Through immunohistochemistry (IHC) we screened several tissue microarrays (TMA) including human, cynomolgusmonkey and macacus rhesus (according to FDA/CE guidelines), rat and mouse normal tissues. Complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) on T-ALL cells were evaluated by flow cytometry. Six different in vivo models on NSG mice (3 with administration of NK-92-CD16+ effectors) have been generated to evaluate mAbs activity in different disease settings: orthotopic, subcutaneous advanced disease (treatments started at 100 mmc) and subcutaneous limited disease (treatments started the day after injection). Results: By screening different cancer cell lines, we observed hUMG1 target to be highly expressed on malignant T-ALL cells. We then tested T-ALL patient-derived blasts from 48 samples (40 pediatric and 8 adults) collected at diagnosis. The antigen was expressed on 23 out of 48 (48%), and most of them belonged to the subset of cortical (EGIL TIII) T-ALL group. By contrast the target antigen was not expressed on normal bone marrow cells from healthy donors. The analysis of the TMA including human normal tissues revealed a specific binding for thymus cortical lymphocytes only, leading us to hypothesize an acceptable safety profile. A humanized mAb, named hUMG1, and an afucosylated version of this mAb (aUMG1) were then developed. By gene expression profiling, western blot and flow cytometry, we observed that target binding by hUMG does not exert any direct activity on neoplastic cells. Subsequently, to investigate CDC, ADCC or ADCP, T-ALL cells were cultured in the presence of complement, peripheral blood mononuclear cells or macrophages, at increasing concentrations of both antibodies. Neither hUMG1 nor aUMG1 were able to induce CDC on target cells. Conversely, both mAbs induced CD16 downregulation, IFN-g production and degranulation on NK cells (more evident with aUMG1) and significant cytotoxicity against both T-ALL cell lines and primary blasts. Additionally, both mAbs induced ADCP. Lastly we observed a mAb-dependent activation of monocytes in the presence of target cells, as demonstrated by the reduction of CD16+ "non-classical" monocytes. Furthermore, we demonstrated potent activity of both mAbs in different T-ALL in vivo models. In an orthotopic model we observed 5 out of 20 treated mice free of disease after 100 days from injection as compared to none of the control group. In both subcutaneous models, we observed a strong ability of our antibody to delay tumor growth and to increase mice survival. Of note, the addition of NK-92-CD16+ strongly improved the activity of aUMG1. In the attempt to bring this antibody from bench to bedside, we assessed, through IHC, the expression of the hUMG1 target in healthy tissues from cynomolgus monkey, macacus rhesus, rat and mouse. We did not observe any reactivity, suggesting that the mAb target is very specific for human cells. Conclusion: Here, we demonstrated that hUMG1 mAb recognizes an antigen specifically expressed on the majority of T-ALL, and its binding is able to mediate effective cytotoxicity against leukemia in vitro and in vivo, indicating that this antibody, in particular the aUMG1 version, may represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting Critical Kinases and Anti-Apoptotic Molecules Overcomes Steroid Resistance in Infant MLL-Rearranged Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3885-3885
Author(s):  
Anne Pieta De Groot ◽  
Yoriko Saito ◽  
Eiryo Kawakami ◽  
Mari Hashimoto ◽  
Yuki Aoki ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Combination Treatment ◽  
Peripheral Blood ◽  
Translational Medicine ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Steroid Resistance ◽  
Hematopoietic Stem ◽  
Pdx Models

Acute lymphoblastic leukemia (ALL) with rearrangement of the mixed-lineage leukemia (MLL) gene frequently affects infants and is associated with a poor prognosis. Standard treatment protocol for infant MLL-rearranged ALL (MLL-ALL) includes glucocorticoids (GCs). However, resistance to GCs remains a major problem. Therefore, it is important to find new treatment strategies that overcome GC resistance in MLL-ALL. To identify novel therapeutic targets to overcome GC resistance in MLL-ALL, we compared transcriptional profiles of normal cord blood CD34+CD38- hematopoietic stem and progenitor cells and GC-resistant infant MLL-ALL leukemia-initiating cells (LICs). We found enrichment of the Src family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathways in the LICs. We hypothesized that activation of these kinases may contribute to GC resistance in MLL-ALL cells. Using our previously developed infant MLL-ALL patient-derived xenograft (PDX) models (Aoki et al., Blood, 2015), we recapitulated human GC-resistance in vivo, characterized by increased hCD45+ chimerism in the peripheral blood after treatment with dexamethasone (% peripheral blood human MLL-ALL at pre-treatment: 34.1±5.2% vs. post treatment 51.3±6.6%, n=26). In order to investigate if inhibition of SFKs and FLT3 can overcome GC-resistance, we treated the MLL-ALL PDX models with our previously developed FLT3-SFK multiple kinase inhibitor RK-20449 (Saito et al., Science Translational Medicine, 2013). Combination treatment with dexamethasone and RK-20449 successfully eliminated human MLL-ALL cells from the peripheral blood (dexamethasone alone: 47.2±7.2% vs. combination: 5.6±2.8% hCD45+ cells, p=6.91E-7), bone marrow (dexamethasone alone: 72.2±4.6% vs. combination: 19.5±4.8% hCD45+ cells, p=1.79E-10), and spleen (dexamethasone alone: 52.6±4.8% vs. combination: 11.7±3.5% hCD45+ cells, p=3.75E-9). In addition, combination treatment eliminated infiltrated MLL-ALL from kidney and liver of the MLL-ALL PDX models. Bcl-2 homology domain 3 (BH3) profiling demonstrated that MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic Bcl-2 protein for their survival. Additional inhibition of Bcl-2 by ABT-199 led to complete elimination of MLL-ALL cells in vitro and in vivo in all MLL-ALL patient cases (Figure1). Taken together, in this study we demonstrated that inhibition of SFKs and FLT3 by RK-20449 overcomes GC-resistance in MLL-ALL. Further, we identified Bcl-2 dependence as a mechanism of treatment resistance in MLL-ALL. Therefore, we believe that the combined inhibition of kinase and anti-apoptotic pathways may lead to effective treatment options for highly resistant infant MLL-ALL. Aoki, et al., Identification of CD34+ and CD34- leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood, 2015: p. 967-980. Saito, Y., et al., A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Science Translational Medicine, 2013: p. 1-15. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting the Notch1 and mTOR pathways in a mouse T-ALL model

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6172-6181 ◽  
Author(s):  
Kathleen Cullion ◽  
Kyle M. Draheim ◽  
Nicole Hermance ◽  
Jennifer Tammam ◽  
Vishva M. Sharma ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Therapeutic Benefit ◽  
Pathway Activation ◽  
Mouse Xenograft Model ◽  
Cell Acute Lymphoblastic Leukemia ◽  
End Stage

Abstract Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with γ-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/− leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.

scholarly journals Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2488-2488 ◽  
Author(s):  
Yana Pikman ◽  
Andrew Furman ◽  
Emily S. Lee ◽  
Andrew E. Place ◽  
Gabriela Alexe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cyclin D3 ◽  
Cycle Arrest ◽  
Cell Acute Lymphoblastic Leukemia

Abstract While significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), approximately 10-20% of newly diagnosed patients will experience either induction failure or relapse. Additionally, fewer than 50% of T-ALL patients who experience a relapse are long-term survivors. New targeted therapies are needed for the treatment of this disease. Multiple lines of evidence point to Cyclin D3/CDK4/6 as a potential therapeutic target in T-ALL. Cyclin D3 (CCND3), a direct target of activated NOTCH1, is upregulated in T-ALL, and CCND3 null animals are refractory to NOTCH1 driven T-ALL. CCND3 binds and activates CDK4/6, and the CCND3-CDK complex phosphorylates the tumor suppressor RB leading to cell cycle progression. Previous studies have demonstrated that CDK4/6 small-molecule inhibition is an effective therapeutic strategy for the treatment of NOTCH1-driven T-ALL mouse models. Using the publicly available Genomics of Drug Sensitivity in Cancer data set, we identified NOTCH1 mutations as a biomarker of response and RB mutations as a biomarker of resistance to the CDK4/6 inhibitor palbociclib. We validated that RB null status predicts resistance to the Novartis CDK4/6 inhibitor LEE011 in a panel of T-ALL cell lines. Interestingly, we identified both NOTCH1 mutant, as well as NOTCH1 wildtype, T-ALL cell lines that were sensitive to LEE011 treatment. Mining of publicly available data revealed that CDK6 is consistently marked by a super-enhancer in T-ALL cell lines, both NOTCH1 mutant and wildtype, suggesting another potential reason for sensitivity to CDK4/6 inhibition in this lineage. Treatment with LEE011 also led to a dose-dependent cell cycle arrest and cell death in T-ALL cells, including MOLT4 (NOTCH1 mutant) and MOLT16 (NOTCH1 wildtype). Combinations of drugs with CDK4/6 inhibitors will likely be critical for the successful translation of this drug class because they generally do not induce cell death. Combinations with cytotoxic chemotherapy are predicted to be antagonistic, however, as most of these drugs rely on rapidly proliferating cells, and CDK4/6 inhibition induces cell cycle arrest. To discover effective, and immediately translatable combination therapies with LEE011 in T-ALL, we performed combination studies of LEE011 with agents standardly used for T-ALL treatment, including corticosteroids, methotrexate, mercaptopurine, asparaginase, vincristine and doxorubicin. Combinations of LEE011 with methotrexate, mercaptopurine, vincristine or asparaginase were antagonistic in T-ALL cell lines while the combination with doxorubicin was additive. Combination treatment of LEE011 with corticosteroids had a synergistic effect on cell viability in MOLT4 and MOLT16 cell lines as measured by excess over Bliss additive and isobologram analyses. This combination also decreased phospho RB signaling, increased cell cycle arrest and induced cell death to a greater degree than either drug alone. LEE011 treatment increased CCND3 protein levels, an effect mitigated by glucocorticoid treatment, one possible mechanism contributing to the observed synergy. Additionally, the combination of LEE011 with everolimus, an mTOR inhibitor, was synergistic in these cell lines. We next extended testing to in vivo models of T-ALL. In a MOLT16 orthotopic mouse model, the combination of LEE011 and everolimus significantly prolonged mouse survival compared to treatment with each individual drug alone. The combination of LEE011 with dexamethasone did not extend survival over treatment with LEE011 alone and dexamethasone was inactive in vivo. Both LEE011 and everolimus had on-target activity in the treated mice as measured by inhibition of peripheral blood phospho-RB and phospho-4EBP1. We then tested the combination of LEE011 with dexamethasone in a second mouse model, a MOLT4 orthotopic model. Here, the combination of LEE011 with dexamethasone was more effective in prolonging survival compared to each treatment alone, supporting a heterogeneous response to the combination of LEE011 with dexamethasone in vivo. We conclude that LEE011 is active in T-ALL, and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation in the clinical setting. Disclosures Kim: Novartis Pharmaceuticals: Employment. Stegmaier:Novartis Pharmaceuticals: Consultancy.

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