Combined Targeting of JAK2 with a Type II JAK2 Inhibitor and mTOR with a TOR Kinase Inhibitor Constitutes Synthetic Activity in JAK2-Driven Ph-like Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2529-2529 ◽  
Author(s):  
Ce Shi ◽  
Lina Han ◽  
Qi Zhang ◽  
Kathryn G. Roberts ◽  
Eugene Park ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Synthetic Activity ◽  
Type I ◽  
Type Ii ◽  
Jak2 Inhibitors

Abstract Background and rationale: Philadelphia chromosome-like acute lymphoblastic leukemia ("Ph-like ALL") is a subtype of high-risk B-precursor ALL (B-ALL), which carries a high risk of relapse with conventional chemotherapy(Roberts et al, N Engl J Med. 2014). Rearrangements in CRLF2, leading to overexpression of cytokine receptor for thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALL and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010;Tasian et al, Blood 2014).In addition,JAK2 fusion proteins, such as PAX5-JAK2 represent a novel class of JAK2-driven cellular transformation in B-ALL (Dagmar et al, Blood 2015). Our prior studies in Ph+ B-ALL established that combining tyrosine kinase inhibitors (TKIs) with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) provides greater anti-leukemia efficacy compared to TKIs in Ph+ ALL (Janeset al, Nat. Med. 2013). In this study, we investigated anti-leukemia efficacy and intracellular signaling networks upon combination of type I or type II JAK2 inhibitors and TOR-KIs in JAK2-driven Ph-like ALL models. Methods. The human B-precursor Ph-like ALL cell lines MUTZ5 (which harborsIGH-CRLF2 translocation and JAK2 R683G mutation), MHH-CALL-4 (IGH-CRLF2 translocation and JAK2 I682F),Reh (ETV6-RUNX1 B-precursor ALL cell line)and mouse Arf-null PAX5-JAK2-MIG + IK6-MIR(IL7-dependent primary Arf-/- pre-B cells expressing the dominant negative Ikaros isoform IK6 with PAX5-JAK2 fusion protein) were studied. Signal transduction inhibitors (STIs): JAK2 type I inhibitor ruxolitinib and type II inhibitor NVP-BBT594 (Andraos et al., Cancer Discovery 2012); allosteric mTOR inhibitor rapamycin or mTOR-KI AZD2014. Effects on intracellular signaling were determined using phospho-flow cytometry and Westernblot analysis. Anti-leukemia effects were quantified using CellTiter-Glo viability assay and annexin V flow cytometry. Results. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (Fig 1D). JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, ERK and S6 activation, yet failed to affect4E-BP1 activation. The TOR-KI AZD2014 but not rapamycin fully inhibited phosphorylation of 4E-BP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth inhibition of Ph-like cells. Combination of ruxolitinib and AZD2014 further inhibited cell proliferation, yet did not induce apoptotic cell death. Recent studies indicate persistence of JAK2-mutated cells upon chronic exposure to type I JAK2 inhibitors, through an adaptive resistance mechanism involving JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2 in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of p-STAT5; in contrast, BBT594 diminished bothp-JAK2 and p-STAT5 in both cell lines. Unexpectedly, BBT594 induced apoptotic cell death in all JAK2-driven Ph-like ALL cell lines MUTZ5, MHH-CALL-4 and Arf-null PAX5-JAK2+IK6, but not in REH cells. Combination of BBT594 with AZD2014 further inhibited phosphorylation of JAK2, AKT, 4E-BP1 and eIF4E, and synergistically induced apoptosis and reduced cell viability in Ph-like ALL cell lines(combination index: MUTZ5, 0.71; MHH-CALL-4, 0.57; Arf-nullPAX5-JAK2+ IK6, 0.81). Of importance, BBT594 and AZD2014 combination induced apoptosis in five JAK2-mutant Ph-like ALL xenograft primary samples. In summary, these results suggest that efficient blockade of JAK2/STAT5 with type II JAK2 inhibitors translates into cell death of mutant JAK2-driven Ph-like ALL cells. Furthermore, concomitant blockade of TORC1 signaling with TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4E-BP1 and causes synthetic activity, providing avenues for novel rationally designed combinatorial regimens in this subset of Ph-like B-ALL. The in vivo studies to test these hypotheses are ongoing using patient-derived xenografts. Disclosures Jabbour: Pfizer: Consultancy, Research Funding. Tasian:Incyte: Consultancy; Gilead: Research Funding. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Dual Targeting of JAK2 Signaling with a Type II JAK2 Inhibitor and of mTOR with a TOR Kinase Inhibitor Induces Apoptosis in CRLF2-Rearranged Ph-like Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3706-3706 ◽  
Author(s):  
Ce Shi ◽  
Lina Han ◽  
Yoko Tabe ◽  
Hong Mu ◽  
Shuo-Chieh Wu ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Mtor Inhibitors ◽  
Type I ◽  
Type Ii ◽  
Jak2 Inhibitor

Abstract Philadelphia chromosome-like acute lymphoblastic leukemia (“Ph-like ALL”) is a subtype of high-risk B-precursor ALL (B-ALL) that carries a high risk of relapse after conventional chemotherapy (Mullighan et al, N Engl J Med. 2009). Rearrangements in CRLF2, leading to overexpression of the receptor for the cytokine thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALLs and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010; Tasian et al, Blood 2014). Previous studies established that combining a tyrosine kinase inhibitor (TKI) with an mTOR inhibitor provides greater anti-leukemia efficacy than a TKI alone in Ph+ B-ALL (Janes et al, Nat. Med. 2013). While allosteric mTOR inhibitors such as rapamycin only partially block mTORC1 and do not directly inhibit mTORC2, second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) efficiently block both mTOR outputs and show greater efficacy when combined with TKIs. In this study, we investigated anti-leukemia efficacy and intracellular signaling networks in Ph-like CRLF2+ ALL models treated with combinations of a type I or type II JAK-2 inhibitor and a TOR-KI. The inhibitors were tested in human B-precursor Ph-like ALL cell lines MUTZ5 (IGH@-CRLF2 translocation, JAK2 R683G mutation) and MHH-CALL-4 (IGH@-CRLF2 translocation, JAK2 I682F mutation), B-ALL cell line REH (CRLF2wt), and primary CRLF2+ xenograft cells in vitro. For signaling and growth inhibition studies, cells were stimulated with 25 ng IL-7 or TSLP for 30 min, then with JAK2 type I inhibitor ruxolitinib (500nM) or type II inhibitor NVP-BBT594 (500nM) (Andraos et al., Cancer Discov. 2012) and allosteric mTOR inhibitor rapamycin or TOR-KI AZD2014. Effects on intracellular signaling were determined by phospho-flow cytometry. Anti-leukemia effects were characterized by viable cell counts and annexin V flow cytometry. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (p-JAK2(Tyr1008), p-STAT5(Ty694)) and AKT/pS6 signaling (p-AKT(Ser473), p-rS6(S235/236) (Fig. 1A). Stimulation with IL-7, mimicking support by the normal bone marrow environment, induced a lesser degree of activation of these phospho-proteins, except for p-4EBP1(T37/46), which was constitutively highly expressed in these cells and further induced by IL-7. These findings warranted combination studies of JAK2 and mTOR inhibitors. JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, and S6 activation, yet failed to decrease p-4EBP1 (Fig. 1A). AZD2014 but not rapamycin fully inhibited p-4EBP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth arrest in CRLF2+ cells (Fig. 1A, C). In turn, combination of ruxolitinib and AZD2014 further reduced cell proliferation but did not induce apoptotic cell death (Fig. 1B, D). Recent studies indicate persistence of JAK2-mutated cells in myeloproliferative neoplasms upon long-term exposure to a type I JAK2 inhibitor, mediated by JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2, in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of STAT5 phosphorylation; in contrast, BBT594 diminished both p-JAK2 and p-STAT5. Unexpectedly, BBT594 induced apoptotic cell death in both MUTZ5, MHH-CALL-4 (Fig 1B) and in ALL blasts recovered from primary CRLF2+ xenograft and grown in OP9 in vitro co-culture; the combination of BBT594 with AZD2014 increased apoptosis and reduced cell viability even further, in both cell lines and in stroma-attached primary ALL cells. In summary, these results suggest that efficient blockade of JAK2/STAT5 with a type II JAK2 inhibitor translates into cell death of JAK2-addicted CRLF2-rearranged cells and may have the capacity to eliminate JAK2-mutated clones. Concomitant blockade of TORC1 signaling with a TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4EBP1 and causes synthetic lethality, providing avenues for novel, rationally designed combinatorial regimens in this subset of Ph-like B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

1994 ◽  
Vol 180 (4) ◽  
pp. 1547-1552 ◽  
Author(s):  
M G Cifone ◽  
R De Maria ◽  
P Roncaioli ◽  
M R Rippo ◽  
M Azuma ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Apoptotic Cell ◽  
Human Tumor ◽  
Apoptotic Cell Death ◽  
Cell Surface Receptor ◽  
Cross Linking ◽  
Cell Extracts

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

scholarly journals Targeting Polo-like Kinase 4 Triggers Polyploidy and Apoptotic Cell Death in TP53-Mutant Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1167-1167
Author(s):  
Edward Ayoub ◽  
Rafael Heinz Montoya ◽  
Vakul Mohanty ◽  
Wencke Walter ◽  
Tallie Patsilevas ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Intellectual Property Rights ◽  
Research Funding ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Advisory Committees ◽  
Grant Support ◽  
Support Research

Abstract Background: TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype, high incidence of minimal residual disease (MRD), and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). While numerous novel treatment regimens, including the combination of the BCL2 inhibitor venetoclax (VEN) and hypomethylating agents (HMA), have emerged as partially effective treatments and resulted in higher remission rates in patients with TP53-mutant AML, full clearance of the mutant TP53 clone is rarely achieved and the majority of patients relapse (Short et al., 2021; Takahashi et al., 2016). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies. Results: In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, and to better understand mechanisms of resistance, we performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations using samples collected from TP53-mutant or TP53-wt high-risk AML patients. Samples were collected at diagnosis (DX) and post-treatment (POSTTX) (total number of samples n= 67, TP53-mutant=36, TP53-wt=31). Diagnostic samples include bulk AML, leukemic stem cells (LSCs), and post-treatment samples including bulk mononuclear cells (MNCS) and patient specific MRD (total n= 67, DX_Bulk=15, DX_LSCs=15, POSTTX_MNCs=14, POSTTX_MRD=23). Differential gene expression analysis of TP53-mutant samples indicates a positive enrichment of the following pathways: G2/M checkpoint, MYC targets, and mitotic spindle, among others. We focused here on genes associated with TP53-mutant AML enriched pathways, and identified a key regulator of centriole biogenesis, one of E2F targets: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples . Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML. To test the rigor of this finding, we interrogated the Munich Leukemia Laboratory (MLL) data base and analyzed their clinically annotated (e.g. karyotype, survival, complete blood counts, previous treatments ... etc) RNA-seq dataset of 726 AML samples (TP53-mutant=72, TP53-wt=654). TP53-mutant AML samples consistently showed significant PLK4 upregulation (p= 0.0003). We analyzed PLK4 expression and its correlation with TP53 mutations in The Cancer Dependency Map project dataset (1375 cell lines in 35 different types of cancers) (p= 0.004). Furthermore, we found significantly higher PLK4 protein levels in TP53-mutant AML MOLM13 cell lines when compared with syngeneic TP53-wt AML MOLM13 cells. Experimentally, we found that PLK4 inhibition using 25nM CFI-400945 in TP53-mutant AML MOLM13 cell lines triggers polyploidy > 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p< 0.0001). Finally, we show that polyploidy is not reversible after drug removal and results in significantly increased levels of apoptotic cell death in TP53-mutant AML MOLM13 cells (Fig.1B). Conclusion: Our data suggest that TP53-mutant AML expresses higher levels of PLK4 in comparison to TP53-wt AML, and targeting PLK4 triggers polyploidy and apoptotic cell death in TP53-mutant AML. A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (Clinical Trial ID: NCT04730258, TWT-202). Figure 1 Figure 1. Disclosures Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Borthakur: Takeda: Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Konopleva: Ascentage: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Sanofi: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Andreeff: Senti-Bio: Consultancy; ONO Pharmaceuticals: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Breast Cancer Research Foundation: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Medicxi: Consultancy; Amgen: Research Funding; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

scholarly journals The NSD2 p.E1099K Mutation in Relapse Pediatric Acute Lymphoblastic Leukemia Is Linked to Mercaptopurine Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3962-3962
Author(s):  
Jason Saliba ◽  
Joanna Pierro ◽  
Nikki Ann Evensen ◽  
Anita Qualls ◽  
Natasha Belsky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Counting ◽  
Type I ◽  
Type Ii ◽  
Set Domain

Abstract Introduction: While the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically, the prognosis for those who relapse remains poor. One of the most common alterations found at relapse is the p.E1099K missense change within the SET domain of NSD2, a histone methyltransferase that di-methylates histone 3 lysine 36 (H3K36). NSD2 has 3 isoforms, two of which, Type II (canonical) and REIIBP (C-terminal), contain the SET domain, and another, Type I (N-terminal), that does not. The p.E1099K mutation leads to increased enzymatic activity, but pathways leading to a clonal advantage are unknown in ALL. Methods: We used short hairpin RNAs (shRNAs) to target knockdown of two combinations of NSD2 isoforms: shI/II targets Types I and II, shII/RE targets Type II and REIIBP. Three different B-cell lines (Reh, 697, and KOPN-8) with 2 wildtype (WT) copies of NSD2 were stably transduced with shII/RE. Two B-Cell lines, RS4;11 and RCH-ACV, heterozygous for the NSD2 p.E1099K mutation, were transduced with shI/II and shII/RE. As a control, each B-cell line was stably transduced with a scrambled non-targeting (NT) shRNA. NSD2 knockdown was confirmed by Western Blots. Cell lines were treated for 5 days with chemotherapy agents commonly used in pediatric ALL treatments (mercaptopurine (MP), cytarabine, methotrexate, prednisone, and doxorubicin). Cytotoxicity was assessed by CellTiter- Glo® and significance between IC50s was determined by ANOVA and post hoc Tukey test. Cell proliferation was measured by cell counting with trypan blue. Cell cycle progression in RS4;11 lines was monitored with Edu staining and flow cytometry with and without exposure to MP. Results: Similar to previously reported results, knockdown of NSD2 in the 3 WT B-cell lines had no effect on cell proliferation. However, shI/II reduced growth by 40% in RS4;11 and 20% in RCH-ACV, while shII/RE decreased proliferation by 45% in RS4;11 and 55% in RCH-ACV when compared to their NT control. In RS4;11, both shI/II and shII/RE led to a similar 10% decrease in cells progressing through S phase compared to NT, which could be due to either a slower progression through cell cycle or less cells entering the cell cycle. Knockdown of NSD2 resulted in sensitivity to 6MP compared to NT in both RS4;11 and RCH-ACV lines. RS4;11 shII/RE had an IC50 3.2-fold more sensitive ( p<.01) and the RS4;11 shI/II IC50 was 1.25-fold more sensitive (NS) versus the NT control. Similarly, RCH-ACV shII/RE had an IC50 3.4-fold more sensitive (p<.01) and the RCH-ACV shI/II IC50 was 2.6-fold more sensitive (p<.01) compared to the NT control. No significant changes in drug sensitivity were noted for the 3 WT NSD2 knockdown B-cell lines compared to their NT controls. During a 120 hour exposure to MP, 34% more RS4;11 shII/RE cells were arrested in the G phase than NT controls, while 26% more RS4;11 shI/II cells were arrested in G phase relative to NT controls. This result indicates MP exposure leads to a reduced percentage of knockdown cells able to progress through the cell cycle. Overall, simultaneously reduced expression of Type II and REIIBP had a greater effect of on cell proliferation and MP response compared to the co-reduction of Types I and II NSD2 in the p.E1099K heterozygous cell lines. Conclusion: The p.E1099K mutation confers a growth advantage and resistance to MP, a cornerstone of ALL therapy. Concurrent reduction of Type II and REIIBP expression by shII/RE resulted in the largest impact on proliferation and MP sensitivity. Both of these isoforms include the SET domain containing the p.E1099K mutation, which indicates one or both isoforms could be responsible for changes in the chromatin state and other possible alterations that lead to a clonal advantage. Based on our findings, determining the mechanism of resistance to MP imparted by NSD2 p.E1099K is now a top priority. Disclosures No relevant conflicts of interest to declare.

scholarly journals BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 280-280 ◽  
Author(s):  
Ernesto Diaz-Flores ◽  
Evan Q. Comeaux ◽  
Kailyn Kim ◽  
Kyle Beckman ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Chromosomal Number ◽  
Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Bad Phosphorylation Is Reduced in Response to Inhibition of Constitutively Activated FLT3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1367-1367
Author(s):  
Kyu-Tae Kim ◽  
Mark Levis ◽  
Donald Small
Keyword(s):  
Cell Death ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Apoptotic Cell ◽  
Myeloid Cell ◽  
Human Leukemia ◽  
Erk Mapk ◽  
Flt3 Inhibitors ◽  
Multiple Signaling

Abstract Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Previously we showed that human leukemia-derived myeloid cell lines having constitutive activation of FLT3 undergo apoptotic cell death when treated with FLT3 inhibitors. Examining the anti- and pro-apoptotic proteins in constitutively activated FLT3 signaling in BaF3/ITD, MOLM-14 and MV4-11 cell lines, we found that the level of Bad phosphorylation was greatly decreased by FLT3 inhibitor treatment. Bad is a proapoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death. Survival factors such as IL-3 inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136. Both Ser112 and Ser136 are rapidly dephosphorylated after treatment with FLT3 inhibitors in BaF3/ITD, MOLM-14, and MV4-11 cells. We also found Bad to be highly phosphorylated in both wild-type and mutant FLT3 primary AML samples. In confirmation of the cell line data, Bad was rapidly dephosphorylated after treatment of the primary samples with lestaurtinib (CEP-701). Upstream molecules responsible for phosphorylation of Bad include Akt, Erk/MAPK, Pim-1, and Pim-2. We previously reported Pim-1 as one of the genes whose expression is induced by constitutively activated FLT3. We and other groups have also shown that constitutively activated FLT3 induces multiple signaling pathways, including PI3/Akt, Erk/MAPK, and Jak/STAT. The dephosphorylation of Bad in response to FLT3 inhibition also correlates with deactivation of Stat5, MEK1/2 and Akt kinase activities and decreased expression of Pim-1. Thus, Bad may be one of the points that these multiple signaling pathways converge in FLT3-mediated cell survival. Thus, it appears that activated FLT3 phosphorylates Bad, resulting in the inability of Bad to bind to Bcl-xL, therefore blocking apoptosis. This is the first report of deactivation of a pro-apoptotic Bcl-2 family member induced by constitutively activated FLT3.

Inhibition of the Human Double Minute (HDM)-2 E3 Ubiquitin Ligase Activates Different Programmed Cell Death (PCD) Pathways in Models of Non-Hodgkin Lymphoma (NHL) with Wild Type (wt) and Mutant (mut) p53

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3618-3618 ◽  
Author(s):  
Richard Julian Jones ◽  
Janine Arts ◽  
Robert Z Orlowski
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Annexin V ◽  
Type I ◽  
Type Ii ◽  
Inhibition Of Proliferation ◽  
Acridine Orange Staining ◽  
The Impact

Abstract Background: The ubiquitin-proteasome pathway has been validated as a target for NHL with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of anti-apoptotic heat shock proteins, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor. Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule JNJ-26854165, an inhibitor of HDM-2-function, in both p53 wt and mut cell line models. Results: Treatment of wt p53 NHL cell lines with JNJ-26854165 induced a dose- and time-dependent inhibition of proliferation, with an IC50 in the 0.02–0.3 μM range. Cell death, which was typically seen within 48 hours of HDM-2 inhibition, occurred through induction of type I PCD, as judged by the appearance of increased staining with Annexin V and activation of caspase 3. While cell lines with mut p53 were generally less sensitive than their wt p53 counterparts, JNJ-26854165 remained potent, with an IC50 in the 0.05–0.6 μM range. The latter cell lines showed a longer kinetics of death, with PCD being seen within 72 hours of drug exposure. Notably, in these mut p53 cell lines, very little Annexin V staining or caspase 3 activation was seen, consistent with a minor role for type I PCD. Instead, mut p53 cell lines demonstrated an increased content of acidic vacuoles by acridine orange staining, increased expression of Beclin 1 and Sequestosome 1/p62, and conversion of microtubule-associated protein 1 light chain 3 form I to form II, consistent with activation of type II PCD, or autophagy. Also, electron microscopy showed an increased presence of autophagosomes and autolysosomes, further supporting the activation of this pathway. Combinations of JNJ-26854165 with other agents, including rapamycin, doxorubicin, and an inhibitor of Bcl 2, showed enhanced anti-proliferative effects in a sequence-dependent fashion, which were greatest when the chemotherapeutic preceded the HDM-2 inhibitor. Combination index analysis revealed that these interactions met criteria for synergy. Conclusions: Inhibition of the function of HDM-2 using JNJ-26854165 is a promising approach that is effective against both wt and mut p53 models by activating type I and type II PCD, respectively. The effectiveness of JNJ-26854165 was enhanced in combination with currently used chemotherapeutics in a sequence specific manner, providing a rationale for translation of this novel approach into the clinic.

scholarly journals D-carvone induced ROS mediated apoptotic cell death in human leukemic cell lines (Molt-4)

2021 ◽  
Vol 17 (1) ◽  
pp. 171-180
Author(s):  
Petchi Iyappan ◽  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Mtt Assay ◽  
Apoptotic Cell ◽  
Biological Activities ◽  
Lymphoblastic Leukemia ◽  
Apoptotic Cell Death ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Intracellular Ros

The immature lymphoid cells with chromosomal structural and numerical abnormalities cause the acute lymphoblastic leukemia (ALL). This hematologic disorder constitutes about 25% of cancer prognosis among children and adolescents. D-Carvone, a monocyclic monoterpene obtained from the essential oils extracted from plants is reported to possess the various biological activities. The present study was aimed to investigate the anticancer potential of D-Carvone against the human leukemic Molt-4 cells. The cytotoxicity of DCarvone was analyzed by MTT assay. The level of lipid peroxidation and antioxidants were determined. The intracellular ROS, MMP and apoptosis were demonstrated by fluorescent staining techniques. The MTT assay revealed that the D-Carvone treatment suppressed the viability of Molt-4 cells and the IC50 was determined at 20 μM/ml. The D-Carvone treatment was increased the oxidative stress and reduced the level of antioxidants in the Molt-4 cell lines. The increased intracellular ROS, apoptotic cell death, and diminished MMP was noted in the D-Carvone treatment. In the Molt-4 cells, D-carvone induced the apoptosis in a time and dose dependent manner by the activation of caspases-8, -9 and -3. Thus, data provide insights for the clinical application of D-Carvone in the treatment of blood cancer Molt-4 cells. Our study suggests the therapeutic potential D-Carvone for the treatment of leukemia in future.

Anti-Leukemia Activity of the Selective BCL-2 Inhibitor ABT-199 in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By MCL-1/BCL-2 Expression Serving As Biomarker for Treatment Response

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1081-1081
Author(s):  
Felix Seyfried ◽  
Salih Demir ◽  
Rebecca Hörl ◽  
Stefan Köhrer ◽  
Annika Scheffold ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
High Ratio ◽  
Cell Precursor ◽  
Free Survival ◽  
Ic50 Values

Abstract Despite superior outcome and survival of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), relapse occurs in 10-20% and is associated with poor outcome, clearly indicating future challenges including reduction of relapse rates and effective treatment of reoccurred leukemia. Deficiencies in cell death and survival pathways have been implicated in therapy failure and treatment resistance in BCP-ALL. Members of the BCL-2 family are key regulators of these pathways and are therefore of interest as therapeutic targets. The small molecule ABT-199 binds selectively to BCL-2, inhibits its anti-apoptotic function and leads to release of pro-apoptotic molecules. Recently, ABT-199 has demonstrated clinical activity, particularly in poor prognosis CLL. However, insensitivity and resistance in different cases clearly emphasize the need of predictive markers for upfront identification of ABT-199 responsive leukemias. Here, we analyzed sensitivity for ABT-199 in a series of individual BCP-ALL samples, addressed mechanisms of resistance and evaluated markers indicating response to ABT-199. Anti-leukemic activities of ABT-199 were investigated in BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primograft samples (n=17), which were established by transplantation of primary patient ALL cells obtained at diagnosis onto NOD/SCID mice. Half maximal inhibitory concentrations (IC50) for ABT-199 were analyzed for each sample. Expression of apoptosis regulating molecules was investigated by western blot analysis and associated with ABT-199 responsiveness. Two MCL-1 deficient ALL cell lines were generated by CRISPR/Cas9 gene editing. Leukemia free-survival of ALL bearing animals was analyzed after in vivoABT-199 treatment. The majority of BCP-ALL samples showed sensitivity for ABT-199 induced cell death in the nanomolar range, both in cell lines (n=4, IC50: 29 - 422 nM) and patient-derived primograft samples (n=10, IC50: 1.7 - 74 nM), while 2 cell lines and 7 primograft leukemias showed insensitivity with IC50 values above 1 µM. ABT-199 binds directly to BCL-2 and upon binding, pro-apoptotic Bcl-2 family molecules like Bim are dislocated from BCL-2 and induce apoptosis. The anti-apoptotic BCL-2 family member MCL-1 is not bound by ABT-199, but sequesters pro-apoptotic molecules dislocated from BCL-2 leading to interruption of apoptosis induction. Therefore, we addressed expression levels of BCL-2 and MCL-1. We found high BCL-2 levels in ABT-199 sensitive and low BCL-2 levels in resistant leukemia samples and an opposite pattern for MCL-1 (high in resistant and low MCL-1 in sensitive ALL), in line with previous reports. Most interestingly, a high ratio of MCL-1 to BCL-2 expression (high MCL-1, low BCL-2) was significantly associated with high IC50 values/resistance (Spearman Rho correlation, p= .01), whereas a low MCL-1/BCL-2 ratio indicated ABT-199 sensitivity. Two of the 6 cell lines showed ABT-199 resistance (IC50 > 1 µM) and high Mcl-1 expression. Effective MCL-1 knock-out in both cell lines led to a clear sensitization for ABT-199 with up to 40-fold reduced IC50 values, clearly indicating MCL-1 as a key mediator of ABT-199 resistance in BCP-ALL. Finally, we also evaluated the anti-leukemia activity of ABT-199 in a preclinical setting in vivo. Two patient-derived leukemias, one with a low MCL-1/BCL-2 ratio of 0.9 and the other with a high ratio of 16.1, indicative of ABT-199 sensitivity or resistance, were transplanted onto NOD/SCID mice and treated with ABT-199 for 10 days after ALL engraftment. Most interestingly, a significantly increased leukemia free survival was observed in ABT-199 as compared to vehicle treated recipients (p<0.001) of the leukemia with the low MCL-1/BCL-2 ratio, in contrast to similar survival times of vehicle or ABT-199 treated animals bearing the high MCL-1/BCL-2 ratio ALL, clearly showing the predictive value of BCL-2 and MCL-1 levels in BCP-ALL. Taken together, ABT-199 shows anti-leukemia activity in the majority of BCP-ALL samples, with a strong association of high BCL-2 and low MCL-1 levels with ABT-199 sensitivity. Silencing of MCL-1 clearly revealed a crucial role for MCL-1 as mediator of ABT-199 resistance. Importantly, in vivo evaluation of ABT-199 in a preclinical setting highlighted the predictive value of BCL-2/MCL-1 expression for the identification of patients who would benefit from future BCL-2 directed therapies. Disclosures Stilgenbauer: Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding.

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