scholarly journals Increased efficacy of histone methyltransferase G9a inhibitors against MYCN-amplified Neuroblastoma

2019 ◽  
Author(s):  
Jacob Bellamy ◽  
Marianna Szemes ◽  
Zsombor Melegh ◽  
Anthony Dallosso ◽  
Madhu Kollareddy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Cancer Therapeutics ◽  
Histone Methyltransferase ◽  
Tumour Suppressor Genes ◽  
Synthetic Lethal ◽  
Ezh2 Expression ◽  
Primary Determinant ◽  
Targeted Inhibition

AbstractTargeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a paediatric solid tumour with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumours is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638 and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumour suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2 and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumours.

scholarly journals Protein kinase Cδ inactivation inhibits cellular proliferation and decreases survival in human neuroendocrine tumors

2011 ◽  
Vol 18 (6) ◽  
pp. 759-771 ◽  
Author(s):  
Zhihong Chen ◽  
Lora W Forman ◽  
Kenneth A Miller ◽  
Brandon English ◽  
Asami Takashima ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroendocrine Tumors ◽  
Small Molecule ◽  
Cellular Proliferation ◽  
Small Molecule Inhibitors ◽  
Cancer Therapeutics ◽  
Therapeutic Modality ◽  
Ras Signaling ◽  
Normal Tissues ◽  
Induced Apoptosis

The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21Rassignaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.

Combination of a MEK Inhibitor, AZD6244, and Dual PI3K/mTOR Inhibitor, NVP-BEZ235: An Effective Therapeutic Strategy for Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3978-3978 ◽  
Author(s):  
Hongtao Liu ◽  
Ernesto Diaz-Flores ◽  
Xavier Poiré ◽  
Olatoyosi Odenike ◽  
Greg Koval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Apoptotic Cell ◽  
Viable Cell ◽  
Apoptotic Cell Death ◽  
Cell Numbers ◽  
Targeted Inhibition

Abstract Abstract 3978 It has been demonstrated that MEK/MAPK and PI3K/Akt are constitutively activated in the majority of AML cases and that their aberrant expression is associated with a poor prognosis. Targeted inhibition of either the MEK/MAPK or the PI3K/Akt pathway alone has only demonstrated mild to modest clinical activity, possibly due to feedback activation of compensatory pathways. Thus, preclinical studies have recently turned to targeted inhibition of both of these pathways simultaneously. In the current study, the efficacy of the combination of two orally available inhibitors to MEK (AZD6244, Astra Zeneca) and PI3K/mTOR (NVP-BEZ235, Novartis) was evaluated in AML cell lines and in primary AML patient samples. In MV 4;11 AML cells (harboring both the MLL re-arrangement and FLT3 internal tandem mutation), AZD6244 or BEZ235 alone moderately decreased viable cell numbers by 30–40% as measured by the MTS assay, a colorimetric assay for cellular growth and survival, but the combination of these two had a dramatic additive effect with a decrease of viable cell numbers by 70–80%. Similar effects were observed in AML cell lines with different cytogenetic and molecular abnormalities including THP-1 [t (6;11)], HL-60, KG-1 [del(5q)], and Kasumi-1 [t(8;21)]. Similar results were also obtained in leukemia cells from 3 patients with AML with different recurring cytogenetic abnormalities. Apoptotic cell death was determined by detection of <2N DNA using 7AAD staining, and the cell cycle was measured using BrdU incorporation followed by flow cytometric analysis. The combination therapy additively induced apoptotic cell death up to 50–60% and cell cycle arrest, whereas either inhibitor alone resulted in only mild apoptotic cell death (∼15-30%). Although dual pathway inhibition was efficacious in all AML cell lines, no additive effect of dual inhibition was observed in Jeko-1, a mantle cell lymphoma cell line. To evaluate the underlying mechanisms of apoptosis, flow cytometry was used to detect phospho-protein and apoptosis-associated proteins. Interestingly, inhibition of MEK/MAPK alone with AZD6244 resulted in decreased pErk level, but increased pmTOR and anti-apoptotic Mcl-1 levels. These results suggest a feedback activation of PI3K/Akt/mTOR pathway, which could be abrogated by the addition of BEZ235. Similarly, inhibition of PI3K/Akt/mTOR resulted in increased pErk and pJNK which could be abrogated by adding AZD6244. AZD6244 also resulted in increased expression of pro-apoptotic Bim, and anti-apoptotic Bcl-2 in AML cell lines, which could not be abrogated by inhibition of PI3K/Akt/mTOR by BEZ235, suggesting that the modulation of these two proteins is independent of the PI3K/Akt/mTOR pathway. Taken together, these findings suggest that inhibition of Bcl-2 might further sensitize AML cells to apoptotic cell death induced by the combination of AZD6244 and BEZ235. In conclusion, these data provide a strong rationale for drug combination targeting of PI3K/Akt/mTOR and MEK pathways for the treatment of AML. Furthermore, inhibition of BCl-2 anti-apoptosis family members may, in part, explain the efficacy of dual signaling blockade in AML cells and suggests an additional therapeutic targeting strategy. Single agent small molecule inhibitors of PI3K/Akt/mTOR, MEK and BCL-2 are already being tested in early phase clinical trials in solid tumors and in hematological malignancies; thus, feasibility studies of combinations of these small molecule inhibitors should be designed to test their efficacy in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Selective Killing of Mixed Lineage Leukemia Cells by a Potent Small-Molecule DOT1L Inhibitor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 780-780
Author(s):  
Roy M Pollock ◽  
Scott R Daigle ◽  
Edward J Olhava ◽  
Carly A Therkelsen ◽  
Christina R Majer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Small Molecule ◽  
Fusion Proteins ◽  
Histone Methyltransferase ◽  
Mixed Lineage Leukemia ◽  
Model Systems ◽  
Targeted Therapeutics ◽  
In Vivo Models ◽  
Mll Gene ◽  
H3k79 Methylation

Abstract Abstract 780 Rearrangements of the mixed lineage leukemia (MLL) gene on chromosome 11q23 are found in over 70 % of infant leukemias, and approximately 10% of adult acute myeloid leukemias (AML). Patients with MLL-rearranged leukemias have aggressive disease with a poor prognosis. Recent studies suggest that DOT1L, a histone methyltransferase that methylates lysine 79 of histone H3 (H3K79), plays a fundamental role in the development and maintenance of this genetically defined subset of leukemia. Rearrangements of the MLL gene result in the expression of MLL-fusion proteins that gain the ability to recruit DOT1L to chromatin. This leads to aberrantly high levels of H3K79 methylation and gene expression at specific genomic loci, including HOXA9 and MEIS1 that are thought to promote leukemogenesis. These findings, together with studies demonstrating a key role for DOT1L in propagating the transforming activity of MLL-fusion proteins in model systems, support the development of inhibitors of this enzyme as targeted therapeutics for patients bearing MLL-rearranged leukemias. To this end, we have used mechanism-guided design to identify EPZ01, the first small molecule DOT1L inhibitor. This compound is a potent and specific inhibitor of DOT1L methyltransferase activity with a Ki of ~ 400 pM in biochemical assays. EPZ01 acts as a competitive inhibitor with the co-factor S-adenosyl-methionine (SAM), and demonstrates greater than 500-fold selectivity for DOT1L over other lysine and arginine histone methyltransferases. Incubation of MLL-rearranged leukemic cell lines with EPZ01 leads to a dramatic decrease in cellular H3K79 methylation but does not affect the methylation of other histone residues, including H3K4, H3K27, H3K36 and H3K9. Analysis of the effects of EPZ01 on the proliferation of a panel of acute lymphoid leukemia (ALL) or AML-derived human MLL-rearranged cell lines including SEMK2, MV4-11, RS4;11, MOLM-13 and THP-1, and non-rearranged leukemia cell lines including HL-60, Jurkat and U937, reveals anti-proliferative activity that is remarkably selective for cell lines bearing the MLL-rearrangement. EC50 values for inhibition of proliferation by EPZ01 are in the nanomolar to low micromolar range for all MLL-rearranged lines tested. In contrast, EPZ01 shows little or no effect on the proliferation of cells lacking an MLL-rearrangement despite an equal decrease in cellular H3K79 methylation. A more detailed analysis of the cellular effects of EPZ01 in MLL-rearranged cell lines reveals that treatment with the inhibitor causes a decrease in mRNA expression of known MLL-fusion target genes including HOXA9 and MEIS1, cell cycle arrest in G0/G1, an increase in expression of differentiation markers in MLL-rearranged AML cells and death by apoptosis. We are currently evaluating the effects of EPZ01 and related compounds in in vivo models of MLL-rearranged leukemia where preliminary results indicate that we are able to achieve inhibition of DOT1L activity. EPZ01 therefore represents the first example of a histone methyltransferase inhibitor that selectively kills tumor cells bearing a defined genetic lesion. These data provide compelling validation for the development of DOT1L inhibitors as targeted therapeutics for MLL-rearranged leukemias and we are currently working towards this goal. Disclosures: Pollock: Epizyme, Inc: Employment. Daigle:Epizyme, Inc: Employment. Olhava:Epizyme, Inc: Employment. Therkelsen:Epizyme, Inc: Employment. Majer:Epizyme, Inc: Employment. Song:Epizyme, Inc: Employment. Allain:Epizyme, Inc: Employment. Sneeringer:Epizyme, Inc: Employment. Johnston:Epizyme, Inc: Employment. Porter Scott:Epizyme, Inc: Employment. Jin:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Chesworth:Epizyme, Inc: Employment. Moyer:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Copeland:Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment.

scholarly journals Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2269-2269
Author(s):  
Trisha Tee ◽  
Titine Ruiter ◽  
Ahmed Dahaoui ◽  
Dorette van Ingen Schenau ◽  
Rico Hagelaar ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Patient Group ◽  
Small Molecule ◽  
Metabolic Activity ◽  
Small Molecule Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Methionine Restriction ◽  
Novel Therapeutic

Abstract Background: MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL. Methods: We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood. Results: We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR. To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect. Conclusions: MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Potential of Aurora Kinases A and B As Therapeutic Targets in Pediatric Acute Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1465-1465
Author(s):  
Stefanie A. Hartsink-Segers ◽  
C. Michel Zwaan ◽  
Carla Exalto ◽  
Mirjam W.J. Luijendijk ◽  
Valerie S. Calvert ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Protein Expression ◽  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Selective Inhibitor ◽  
Growth Delay ◽  
Aurora Kinases ◽  
Cell Counts

Abstract Abstract 1465 Aurora kinases (AURK) A and B play distinct and important roles during mitosis, and many human cancer types are characterized by upregulated AURK expression. Several small-molecule inhibitors targeting AURKA, B or both, are in development and have shown promising anti-tumour activity in vitro and in vivo. However, most studies address the efficacy of these small-molecule inhibitors in adult cancer. Of all childhood cancers, acute leukemia is the most common type and there is a great medical need to improve outcome and side-effects of current treatment strategies. We therefore analyzed the effects of targeting AURKA and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Affymetrix microarray analysis of pediatric ALL (n=297) and AML cases (n=237), and normal bone marrow (nBM) samples (n=8), showed that AURKA and B gene expression was low in all patients. AURK protein expression was determined by western blot and reverse phase protein array. Protein levels of both kinases were significantly elevated in ALL and AML compared to nBM (p<0.0002), especially in patients with an E2A-PBX1 translocation (p<0.002). We used short hairpin RNAs and LNA-based mRNA antagonists to silence AURKA and B expression in three ALL and three AML cell lines of different genetic subtypes. Silencing of AURKA caused no or only minor growth delay, whereas AURKB knockdown resulted in proliferation arrest and apoptosis, as indicated by reduced cell counts and the presence of cleaved PARP. This suggests a pivotal role for AURKB in the survival and proliferation of leukemic cells. A panel of leukemic cell lines was exposed to several AURK-inhibitors, and cell viability was determined with an MTS assay. Comparable sensitivity in the low- to mid-nanomolar range was observed for the pan-Aurora inhibitor VX-680, AURKA-selective inhibitor MLN8237 and AURKB-selective inhibitor Barasertib-HQPA (AZD1152-HQPA), whereas cell lines were relatively resistant to the pan-Aurora inhibitor Danusertib. Since silencing experiments suggested that AURKB would be a suitable target, we tested the efficacy of Barasertib-HQPA in primary ALL cells. ALL patient cells with a high AURKB protein expression, especially E2A-PBX1-positive cases, were more sensitive to Barasertib-HQPA than those with a low expression (p<0.05), whereas nBM cells were resistant up to a concentration of 20μM. In conclusion, inhibition of AURKB, more than AURKA, has an anti-proliferative and pro-apoptotic effect on ALL and AML cells, suggesting that particularly targeting AURKB may be of benefit in the treatment of pediatric acute leukemia. Moreover, this study shows the potential of both LNA-containing mRNA antagonists and Barasertib, for which clear responses have been observed in adult AML early clinical trials, in the treatment of children with ALL and AML. Disclosures: Hedtjärn: Santaris Pharma: Employment. Hansen:Santaris Pharma: Employment. Koch:Santaris Pharma: Employment.

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