Tyrosine Kinase Inhibitor Screen Identifies the Multikinase Inhibitor, Foretinib, As a Sensitisor of Treatment-resistant B-Precursor ALL to Core Chemotherapeutic Agents

Abstract Abstract 1509 Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood with resistance or relapse occurring in up to 20% of patients. The precise mechanisms of resistance to conventional therapy leading to relapse have not been elucidated. Deregulation of tyrosine kinases (TKs) have been implicated in resistant solid tumours and the of aetiology haemopoietic tumours, Philadelphia – chromosome positive ALL (Ph+ ALL), FLT3 in MLL+ infant ALL and FLT3-ITD subset in acute myeloid leukaemia. The role of TK inhibitors (TKIs) has not been extensively investigated in non-Ph + ALL. We screened 5 B-cell precursor ALL cell lines and 20 primary samples with a library of 34 TKIs. Nalm 6 (t(5;12)), Nalm 17 (normal karyotype), REH (t(12;21)), SD1 and Sup15 (Ph+ ALL) and primary cells were tested at 1μM and 10μM and alterations in cell viability assessed with the Promega CellTiter-Glo assay. A drug was considered to be effective if it induced >50% reduction in cell viability at 1μM. While we demonstrated significant heterogeneity in response to many of the TKIs, we observed reduction in viability to lestaurtinib (FLT3/JAK2), dovitinib (FLT3/FGFR/PDGFR/VEGFR) and bosutinib (Abl/Src) in all cell lines. Compared with Nalm 6 and Nalm 17 which only exhibited sensitivity to these 3 TKIs, REH demonstrated additional sensitivity to crizotinib (ALK/Met) and the quinazoline pan-EGFR inhibitors, afatinib and canertinib. The Ph+ cell lines SupB15 and SD1 responded to the highest number of TKIs, 12 and 14 respectively. These included the expected Bcr/Abl and Aurora kinase inhibitors. Activity of the putative PDGFR/VEGFR TKIs axitinib, linifanib, vargatef and also foretinib (MET/VEGFR2/FLT3) appeared limited to Ph+ cell lines. The cell lines, REH and SD-1, which are resistant to ionizing radiation–induced apoptosis, were selectively inhibited by both the quinazolines. Baseline mRNA expression of the ErbB family was present in all cell lines and therefore did not correlate with response. TKIs inducing the greatest reduction in cell viability across the cell lines were those that target class III/IV/V RTKs. Although all cell lines expressed FLT3 mRNA, reduction in cell viability was not universally induced by the specific FLT3 inhibitor tandutinib at doses of up to 10μM. As observed in previous studies, the level of mRNA transcript did not predict or directly correlate with the response to TKI. A panel of 20 primary ALL samples, representative of common biological features, were screened. We found no correlation between cytogenetics, age, white cell count, post – induction MRD status and response to TKI groups or individual inhibitor. Only 5/20 did not respond to any of the tested TKIs. Lestaurtinib, dovitinib and foretinib reduced cell viability in 7/20 primary ALLs. In addition, canertinib reduced cell viabililty in 6/20 primary ALL samples, afatinib and TAE684 (ALK/MET) both in 5/20 ALL samples respectively and vargatef in 4/20 samples. Based on our preliminary screen, the multikinase inhibitor foretinib was selected as one of several promising candidates for further pre-clinical testing. Recent adult phase 1 solid tumor trials have shown limited toxicity and good bioavailability. Foretinib inhibited leukaemia proliferation with LD50 in nanomolar and low micromolar range; SupB15 (333nM ±49), SD-1 (381nM ±239), Nalm 17 (484nM ±124), REH (689nM ±92) and Nalm 6 (1.84μM ±0.25). Annexin/PI staining, DNA fragmentation and PARP protein cleavage confirmed that the mechanism of cell death was apoptosis. We next investigated whether foretinib could sensitise ALL cell lines to dexamethasone, cytarabine, methotrexate, doxorubicin or mitoxantrone. Drug interactions were modelled using the Biosoft Calcusyn software package. We found that the addition of foretinib resulted in predominantly synergistic interactions in all cell lines (CI<1). The most striking example of synergism was in the dexamethasone-resistant cell line, REH. Addition of a sub–LD50 dose of foretinib led to >50% reduction in cell viability when combined with 1nM dexamethasone compared with no response at 10μM dexamethasone alone. Overall these data support further exploration of TKIs as potential therapeutic agents in childhood ALL. Specifically, we are currently investigating the direct anti-leukemic activity of foretinib in childhood ALL and its synergistic activity with dexamethasone in vivo using our NOG mouse primograft model for ALL. Disclosures: No relevant conflicts of interest to declare.

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Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽

10.1182/blood.v116.21.4469.4469 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4469-4469

Author(s):

Hilmar Quentmeier ◽

Sonja Eberth ◽

Julia Romani ◽

Margarete Zaborski ◽

Hans G. Drexler

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemia Cell ◽

Resistant Cell ◽

Imatinib Resistance ◽

Imatinib Resistant ◽

Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

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Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

Blood ◽

10.1182/blood.v108.11.1384.1384 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1384-1384 ◽

Cited By ~ 3

Author(s):

Russell R. Hoover ◽

Matthew W. Harding

Keyword(s):

Clinical Trials ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Single Agent ◽

Leukemia Cell ◽

Parp Cleavage ◽

Synergistic Activity ◽

Dependent Manner ◽

Wild Type

Abstract MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.

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Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽

10.1182/blood.v120.21.4010.4010 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4010-4010

Author(s):

Liesbeth Bieghs ◽

Ken Maes ◽

Els Van Valckenborgh ◽

Eline Menu ◽

Hans Erik Johnsen ◽

...

Keyword(s):

Growth Factors ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Differential Sensitivity ◽

Expression Ratio ◽

Therapy Strategy ◽

Induced Apoptosis ◽

Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

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FOXM1 Is Overexpressed in B-Acute Lymphoblastic Leukemia (B-All) and Its Inhibition Sensitizes B-All Cells to Chemotherapeutic Drugs

Blood ◽

10.1182/blood.v124.21.2245.2245 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2245-2245 ◽

Cited By ~ 1

Author(s):

Francesca Consolaro ◽

Giuseppe Basso ◽

Giampietro Viola ◽

Eric W-.F. Lam

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Resistant Cell ◽

Chemotherapeutic Agents ◽

Chemotherapeutic Drugs ◽

Glucocorticoid Treatment

Abstract B-lymphoblastic leukaemic (B-ALL) patients that respond poorly to glucocorticoid therapy are predicted to relapse. An understanding of the biological mechanism underlying this poor responsiveness is therefore crucial for the development of more effective diagnostics and therapies. Forkhead box protein M1 (FOXM1) is a key transcriptional factor that regulates the expression of several genes that promote cell cycle progression, proliferation, DNA repair. Its expression is up-regulated in most cancer cells and is often linked to high proliferation rates and poor responsiveness to the therapy. In this context, we studied the role of FOXM1 in B-lymphoblastic leukaemia (B-ALL) in order to understand if FOXM1 could be a key target for leukaemia therapy. Our results showed that FOXM1 expression is higher in both B-ALL patients and cell lines compared to PBMC or CD19+ cells from healthy donors (Figure 1 A, 1 B). Figure 1A: FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1A:. FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1B Figure 1B. Furthermore FOXM1 protein levels were higher in glucocorticoid-resistant cell lines (REH, MHH-CALL2, SEM) when compared to their glucocorticoid-sensitive counterparts (RS4;11, NALM-6), suggesting that FOXM1 may have a role in mediating chemotherapeutic drug sensitivity and resistance in B-ALL. Furthermore, depletion of FOXM1 activity in B-ALL cell lines by either transient knockdown or treatment with a FOXM1 inhibitor, thiostrepton, significantly decreases the cell viability of cells that poorly respond to glucocorticoid treatment (REH). The decrease of cell viability was accompanied by an induction of G2/M arrest of the cell cycle along with a reduction of the S phase. Moreover thiostrepton synergises with common chemotherapeutic agents used in B-ALL therapy increasing their efficiency and overcoming drug resistance. All this data suggest that FOXM1 could be an important therapeutic target for overcoming the resistance to the conventional chemotherapeutic drugs. Disclosures No relevant conflicts of interest to declare.

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A Novel and Highly Selective Dual PI3K/mTOR Kinase Inhibitor VS-5584, Shows Promising Therapeutic Potential For The Treatment Of Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.4433.4433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4433-4433

Author(s):

Nurulhuda Mustafa ◽

Stefan Hart ◽

Wee-Joo Chng

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Mononuclear Cells ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Synergistic Activity ◽

Peripheral Blood Mononuclear ◽

Wide Range

Currently, most multiple myeloma (MM) patients experience relapse and develop resistance to standard treatments. A recent study showed that patients who relapsed had poor outcomes, with an overall survival of only 6 months and an event-free survival of 1 month. The PI3K/mTOR/AKT pathway represents a critical target in MM because it stimulates proliferation, survival, and drug resistance of MM cells. VS-5584 is a novel agent, with specific and equipotent activity against mTOR and all 4 Class I PI3K isoforms, without relevant activity on 400 other lipid and protein kinases. Here we report that VS-5584 is highly efficacious against a wide panel of MM cells including Velcade- and Doxorubucin- resistant cell lines. This efficacy is maintained even in the presence of additional MM growth factors, IL-6 and IGF-1, and seems independent of PTEN status in the cell lines. Importantly, VS-5584 shows similar efficacy in patient myeloma cells and preferential tumor cell targeting compared to healthy peripheral blood mononuclear cells. Further testing in a myeloma xenograft mouse model further confirmed the potency of this compound in vivo. We have also observed synergistic activity in combination with both MM clinical therapeutic Dexamethasone, and novel anti-MM candidate Panabinostat. Comparing the basal expression profile of hypersensitive (H929) vs less sensitive (OPM2, U266) cell lines have identified the interferon alpha/beta pathway as a marker for association with sensitivity. Just recently, VS-5584 has been reported to evidence very favourable pharmaceutical and pharmacological properties in a wide range of solid tumors resistant to standard care therapies. Taken together with our data, this offers a compelling rationale for its clinical development as a single or combination therapy in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

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Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

Pharmaceuticals ◽

10.3390/ph14070682 ◽

2021 ◽

Vol 14 (7) ◽

pp. 682

Author(s):

Jianling Bi ◽

Garima Dixit ◽

Yuping Zhang ◽

Eric J. Devor ◽

Haley A. Losh ◽

...

Keyword(s):

Cell Cycle ◽

Endometrial Cancer ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽

10.1182/blood-2019-126910 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3758-3758

Author(s):

Jianping Li ◽

Catalina Troche ◽

Julia Hlavka Zhang ◽

Jonathan Shrimp ◽

Jacob S. Roth ◽

...

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Cell Lines ◽

Mutant Allele ◽

Gene Editing ◽

Conflicts Of Interest ◽

Chemotherapeutic Agents ◽

Rna Seq ◽

Mesenchymal Transition ◽

Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

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Repurposing dasatinib for diffuse large B cell lymphoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905239116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 16981-16986 ◽

Cited By ~ 5

Author(s):

Claudio Scuoppo ◽

Jiguang Wang ◽

Mirjana Persaud ◽

Sandeep K. Mittan ◽

Katia Basso ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Kinase Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Multikinase Inhibitor ◽

Large B Cell Lymphoma ◽

Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

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Transcriptional Repression of DAPK1 Characterizes a Resistant Phenotype of AML Enforced by Flt3 Signaling and Exclusive Nuclear Abundance of Non-Canonical NFkB2/p52: Synergistic Activity for Flt3 Inhibition along with HDAC Inhibition, or by NFkB Inhibition, for Potentiating ER Stress Apoptosis.

Blood ◽

10.1182/blood.v114.22.2387.2387 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2387-2387

Author(s):

Rajasubramaniam Shanmugam ◽

Annique Wilson-Weekes ◽

Hamid Sayar ◽

Attaya Suvannasankha ◽

Angelo A. Cardoso ◽

...

Keyword(s):

Er Stress ◽

Hdac Inhibitor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Tritiated Thymidine ◽

Transcriptional Activator ◽

Pcr Analysis ◽

Synergistic Activity ◽

Inhibition Of Proliferation ◽

Poor Risk

Abstract Abstract 2387 Poster Board II-364 Treatment failure in AML has been linked with the consequences of Flt3 signaling, although effectors of such resistance are uncertain. However, poor prognostic Flt3-ITD-containing AML's and some other intermediate prognostic groups have been found to have blunted spontaneous unfolded protein response/ER stress apoptotic pathway (Schardt, et al. Clin Canc Res. 15: 3834, 2009) that correlates with adverse outcome of therapy. We investigated the origins of suppressed ER-stress apoptosis in poor-risk AML by assaying a rate-limiting enzyme effector, DAPK1. Real-time RT-PCR analysis of DAPK1 expression, as well as its putative transcriptional activator(s), including c-jun, and its transcriptional repressor(s), including relB, and a gene-set enrichment array (GSEA) of NFkB- and c-jun-responsive (cytokine) genes, was undertaken in 30 AML samples. This included a control cohort of good-prognosis CBF+ve patients. A group of poor prognosis patients, especially those with normal karyotype and Flt3 (ITD) mutation, and those with MLL translocations, had on-average 10-fold (range 2-100-fold) reduction of DAPK1 transcripts, when normalized to expression of c-jun, a transcriptional activator of DAPK1, or to Meis1, another c-jun transcriptional target, which is a validated AML adverse prognostic array gene. Indeed, NK Flt3-ITD or t-MLL AML blasts with DAPK1 repression were characterized by exclusive nuclear presence of p52NFkB/relB, known to attract epigenetic and repressive components, including histone deacetylase enzymes (HDAC's) to the tandem CRE/NFkB sites (“integrated circuit”) at 5' DAPK1 -177bp and -134bp, respectively (Puto and Reed, Genes&Dev 22:998, 2008; Huang et al. Mol Cell 35: 48, 2009; Gade et al. Mol Cell Biol. 28: 2528, 2008). In the t-MLL/Flt3-ITD AML cell line MV-4-11, knockdown of NIK, IKK1, or p52NFkB2, while sparing Flt3-to-JNK/c-jun signaling, sharply upregulated (3-5-fold) DAPK1 expression. Flt3 inhibition in primary AML cells by using R406 (Rigel Pharmaceuticals, also a lead compound in CLL/NHL) a selective tyrosine kinase inhibitor that affects Flt3 auto-phosphorylation (and syk with similar cellular IC50's) also de-repressed DAPK1 expression in AML blasts. This outcome was associated with an inhibition of NIK and p52NFkB (both were also inhibited by Flt3 knockdown in MV-4-11) and a rise in apoptosis in R406-treated primary AML cells occurred. The combination of R406 with an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA) showed synergistic inhibition of proliferation (tritiated thymidine incorporation). ER stress pathway activation evidenced by IREalpha and p-eIF2alpha upregulation was enhanced in primary AML blasts. Bortezomib (a proteasome-targeted inhibitor of NFkB/ER stress potentiator) also strongly inhibited p52NFkB activity and induced apoptosis. These studies reveal that transcriptional/epigenetic suppression of DAPK1 in poor-risk AML is enforced by a Flt3-to-NIK-to-p52NFkB2/relB signal cascade, and support a causative influence on clinical outcomes. Thus, resistance is subject to inactivation by a Flt3-selective TKI, in combination with HDAC inhibitor/or by proteasome-NFkB inhibitor with consequent ER stress apoptosis. A potential subsidiary role for syk inhibition downstream from Flt3 also exists. Taken together, these maneuvers reverse the resistance to apoptosis of NK Flt3-ITD or tMLL primary AML blasts, and including others with over-expressed/active Flt3 wild-type. Disclosures: No relevant conflicts of interest to declare.

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The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1209.1209 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1209-1209

Author(s):

Chun Shik Park ◽

Koramit Suppipat ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

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