scholarly journals Targeting Hypersumoylation in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4060-4060 ◽  
Author(s):  
Walter Hanel ◽  
Liudmyla Tsyba ◽  
Dennis Huszar ◽  
Alex Prouty ◽  
Xiaoli Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Human B Cells ◽  
Mantle Cell ◽  
Normal Human ◽  
Protein Sumoylation

Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphoma (NHL) characterized by genetic dysregulation of CyclinD1. Despite the improvement in response rates with current therapies, MCL patients inevitably relapse and outcomes remain poor. This is particularly true for MCL patients progressing on novel targeted therapies such as ibrutinib, highlighting the continued need for new therapeutic approaches. SUMOylation is a post-translational modification regulated by SUMO Activating Enzymes 1 and 2 (SAE1/2) affecting function, stability, and subcellular localization of a multitude of proteins such as Cyclin D1 and regulating multiple cellular functions such as cell cycle control and DNA damage response. While not yet explored in MCL, it is known that hyper-SUMOylation is associated with augmented cell proliferation and tumor growth of a number of cancers including B-cell NHL. We evaluated the expression levels of SAE1/2, total SUMO1, and SUMO 2/3 in normal human B cells, primary MCL patient samples, and a panel of 8 MCL cell lines via immunoblotting. We found significantly increased levels of SAE1/2 and total protein SUMOylation in 4 out of 5 MCL patient samples and all MCL cell lines compared to normal human B-cells. To validate the SAE complex as a potential therapeutic target in MCL, we performed genetic knockdown of SAE1 and SAE2 using both shRNA and an inducible CRISPR/Cas9 system and found significant reduction in viability of MCL cells (p < 0.001) thus confirming that SUMOylation is essential for MCL survival. TAK-981 (Takeda Pharmaceuticals) is a potent and selective inhibitor of the SAE1/2 complex currently in a phase 1 clinical trial (NCT036483). We found that treatment of MCL cell lines with TAK-981 resulted in time- and dose-dependent cell death in 7 of 8 MCL cell lines (IC50 17 - 62.5 nM at 72 hr) which was associated with relevant decrease in protein sumoylation. MCL cells were sensitive to TAK regardless of ATM or p53 mutations. Finally, TAK-981 treatment prolonged the survival of SCID mice engrafted with a human MCL cell line (Jeko) compared with placebo control [median overall survival (OS): TAK-981, 34 days; placebo, 29 days, p = 0.008] and also extended the survival of a novel patient derived xenograft (PDX) mouse model of ibrutinib-resistant MCL (median OS: TAK-981, 60 days; placebo, 55 days, p = 0.001), thus establishing the in vivo efficacy of TAK-981 in models of aggressive MCL. Mechanistically, 24 hours of treatment with TAK-981 resulted in a profound G2M cell cycle arrest in 6 out of 7 TAK-981-sensitive MCL cell lines. Cell synchronization with palbociclib followed by release into TAK-981 showed significant apoptosis upon G2M re-entry. In addition, in p53-deficient MCL cell lines, we found rapid accumulation of polyploid and aneuploid cells followed by rapid cell death following 48 hours of drug exposure. These findings strongly support mitotic catastrophe as a significant mechanism of tumor cell death mediated by TAK-981. Upon fractionation of cells at distinct phases of the cell cycle, we found significantly increased levels of protein SUMOylation by both SUMO1 and SUMO2/3 at the G2M transition. Further mechanistic data will be presented at the meeting. Given the multiple immune dampening mechanisms of SUMOylation, we are currently studying the anti-MCL immune effects of TAK-981. To do this, we are employing a novel immunocompetent mouse model of MCL in which murine lymphoma cells from Eμ-SOX11/CCND1 double transgenic animals are adoptively transferred into syngeneic mice. These mice develop a systemic lymphoma with morphological, molecular, and phenotypic features characteristic of MCL resulting in death within 3-4 weeks. Preliminary results with this model show that treatment with TAK-981 leads to decrease in lymphoma burden and significant prolongation of survival. Studies into the immune mediated anti-lymphoma effects of TAK-981 using this model are ongoing and will be presented at the meeting. Together, our data strongly support further development of TAK-981 as a novel MCL therapeutic. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Parekh:Karyopharm Inc.: Research Funding; Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding. Maddocks:BMS: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Merck: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees. Baiocchi:Prelude: Consultancy.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1741-1741
Author(s):  
Steffen Klippel ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Douglas McMillin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cancer Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Energy Production ◽  
Advisory Committees ◽  
Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

scholarly journals The Novel Multitarget Small-Molecule Inhibitor SRX3177 Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2262-2262
Author(s):  
Dhananjaya Pal ◽  
Cody C. McHale ◽  
Samon Benrashid ◽  
Poornima Gourabathini ◽  
Krishnaiah Maddeboina ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Molecule Inhibitor ◽  
Mantle Cell ◽  
Ibrutinib Resistance

Abstract Mantle cell lymphoma (MCL) is an aggressive, rare, and difficult to treat subtype of non-Hodgkin's Lymphoma (NHL) that accounts for about 6% of all cases. Although there is no defined standard of care for MCL treatment, some combination of chemo-immunotherapy and rituximab maintenance with or without autologous stem cell transplantation is generally employed depending on the age and fitness of the patient. Despite recent development of novel therapeutics, there is inevitable disease relapse with progressively declining efficacy and increasing frequency of resistance with single agent targeted therapy. Here, we describe the novel multitarget inhibitor SRX3177 which simultaneously hits three oncogenic targets: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic reader protein BRD4. This in silico designed, thieno-pyranone (TP) scaffold-based small molecule inhibitor orthogonally disrupt three targets within the cancer cell with one agent. Targeting the cell cycle with small molecule inhibitors represents a reasonable attempt to treat MCL, as cell cycle-associated genes like ATM, TP53, CDKN2A, CCND1 and CDK4/6 are most frequently mutated in patients. Palbociclib is a well-known single agent CDK4/6 inhibitor that has been employed in both solid and hematological malignancies. Due to its cytostatic nature, treatment with single agent palbociclib often results in the emergence of treatment-resistant clones. Therefore, a combination strategy would theoretically be more effective and can overcome the development of resistance. Moreover, prolonged G1 arrest by CDK4/6 inhibition sensitizes lymphoma cells to PI3K inhibition, suggesting a synthetic lethality relationship between these two agents. Inhibiting the chromatin reader protein BRD4 causes downregulation of target genes c-MYC and BCL2, further increasing cytotoxic capabilities. Hence, we developed SRX3177 as a potent CDK4/6/PI3K/BRD4 triple inhibitor to synergistically inhibit cell cycle progression and induce cancer cell apoptosis. SRX3177 is an ATP competitive CDK4/6 inhibitor (IC 50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC 50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC 50: BD1 = 32.9 nM, BD2 = 88.8 nM). We have tested the efficacy of SRX3177 against a panel of MCL cell lines and report that SRX3177 induces a strong antiproliferative activity with maximal IC 50 0f 340 nM in JeKo-1, 29 nM in Mino cells, and 630 nM for Rec-1 cells while IC 50 values for cell lines Granta and JVM-2 were 1.3 µM and 1.5 µM, respectively. Further, we show that SRX3177 is more potent to tumor cells than the individual PI3K (BKM120), BTK (Ibrutinib), BRD4 (JQ1), and CDK4/6 (palbociclib) inhibitors, and dual PI3K/BRD4 inhibitor SF2523 (backbone for SRX3177) in JeKo-1 cells. Next, we examine the cytotoxic effect of SRX3177 in ibrutinib/palbociclib resistant primary MCL cells. Our results show that SRX3177 triggers cytotoxic response at 500 nM and 1000 nM as compared to the lack of cytotoxicity of combination Ibrutinib and palbociclib at 150 nM and 1000 nM (Fig 1). SRX3177 induces a strong apoptotic response and cell cycle arrest in JeKo-1 and Mino cells at 24hrs. Annexin V/7AAD apoptosis staining confirmed the induction of PCD by SRX3177with increase in c-PARP. Western blot analysis shows SRX3177 treatment blocks both PI3K/AKT signaling and Rb phosphorylation. Moreover, analysis by chromatin immunoprecipitation revealed that SRX3177 effectively blocked BRD4 binding to both the promoter and enhancer of c-MYC (p≤0.01 and p≤0.001) and BCL2 (p≤0.05). SRX3177 also suppresses the c-MYC and BCL2 transcriptional program in both a time- and dose-dependent manner. Our findings also demonstrate a SRX3177-dependent reduction in c-MYC half-life via induction of proteasomal-mediated degradation. This degradation is associated with decreased phosphorylation of c-MYC at Ser62 and increased phosphorylation of c-MYC at Thr58 - indicative of differential regulation of c-MYC stability. Finally, we show that SRX3177 overcomes chronic ibrutinib resistance in Jeko-1 cells with a maximal IC 50 of 150 nM as compared to 64 µM with ibrutinib. Hence, the triple inhibitor SRX3177 has superior potency to ibrutinib in MCL cell lines and succeeds in overcoming ibrutinib-resistance at nanomolar doses. Taken together, our data supports the development of SRX3177 as a novel therapeutic agent for treatment of MCL. Figure 1 Figure 1. Disclosures Martin: ADCT: Consultancy. Park: Takeda: Research Funding; G1 Therapeutics: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company.

scholarly journals Characterization of Mechanisms Underlying Acquired Venetoclax-Resistance in Mantle Cell Lymphoma: BDA-366 As a Potential Treatment Option

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1580-1580
Author(s):  
Daniela Steinbrecher ◽  
Felix Seyfried ◽  
Johannes Bloehdorn ◽  
Billy Michael Chelliah Jebaraj ◽  
Lüder Hinrich Meyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Apoptotic Protein ◽  
Mantle Cell ◽  
Resistant Cells

Abstract In many cancers the equilibrium of pro- versus anti-apoptotic BCL-2 proteins is deregulated. BCL-2 inhibitors like Venetoclax (VEN) have been shown to be highly active drugs in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Despite being highly efficient in cell killing, resistance to VEN can be acquired over time. In addition to understanding the underlying mechanisms of resistance to VEN it is important to identify additional treatment options. BDA-366 is a BCL-2 inhibitor with a different mode of action than the BH3 mimetic VEN. BDA-366 acts by inhibiting the BH4 domain and thereby inducing a conversion of anti-apoptotic BCL-2 into a pro-apoptotic protein. BDA-366 showed high effectivity in inducing apoptosis in CLL cells, in primary as well as in cell lines, while all of the CLL cell lines (n=7) tested were resistant to VEN. Furthermore all of the MCL cell lines (n=5) tested were sensitive to the treatment with BDA-366 while only a subset (3 out of 5) responded to treatment with VEN. In order to investigate whether BDA-366 would be a treatment option for VEN-resistant patients, we generated VEN-resistant MCL cell lines (MINO and MAVER-1) by chronic exposure to the drug. In the resistant cell lines, BCL-2 protein levels were not deregulated. In variance to previous reports in diffuse large B cell lymphoma (DLBCL) (Choudhary et al, Cell Death Dis 2015), resistance in MCL cell lines was not mediated by MCL-1 upregulation. In VEN-resistant MINO cells, MCL-1 expression was similar to the parental cells, while MCL-1 was significantly downregulated in VEN-resistant MAVER-1 cells. In contrast, VEN-resistant MCL cell lines showed BCL-XL upregulation as compared to parental cells, which is in line with results obtained in DLBCL (Choudhary et al, Cell Death Dis 2015). Furthermore, dynamic BH3 profiling validated a dependency on BCL-XL in resistant cells and confirmed that resistance was not mediated by MCL-1. The significance of BCL-XL in mediating resistance to VEN was underlined by additional experiments using navitoclax. In contrast to VEN, navitoclax inhibits BCL-2, BCL-XL and BCL-W and was sufficient to induce apoptosis in both parental and resistant cells. In contrast to the BH3 domain inhibitor VEN, the BCL-2 inhibitor BDA-366 acts by converting BCL-2 into a pro-apoptotic molecule. BDA-366 efficiently induced dose dependent apoptosis in VEN-resistant cells. MINO as well as MINO VEN-resistant cells showed the same sensitivity to BDA-366 while VEN-resistant MAVER-1 cells showed reduced sensitivity to BDA-366 as compared to the parental cells. However, with increased BDA-366 concentrations efficient cell killing was achieved in the VEN-resistant cell lines Overall, these results suggest that VEN-resistance is mostly mediated by permanent upregulation of BCL-XL. BCL-2 levels are not deregulated upon development of resistance to VEN. The inhibition of the BH4 domain and thereby converting BCL-2 into a pro-apoptotic protein proved to be a promising therapeutic option even in cancers with acquired resistance to VEN. Disclosures Döhner: Pfizer: Research Funding; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Pfizer: Research Funding; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1556-1556
Author(s):  
Albert Perez-Ladaga ◽  
Bennett Caughey ◽  
Huafeng Xie ◽  
Stuart H. Orkin ◽  
David B. Sykes ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Resting Cells ◽  
Surface Markers ◽  
Advisory Committees ◽  
Spontaneous Cell Death

Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Mao Yu Peng ◽  
Yasmin Abaza ◽  
Martina Mcdermott ◽  
Monica Mead ◽  
Dennis J. Slamon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Synergistic Effect ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
Single Agent ◽  
Therapeutic Strategies ◽  
Private Company ◽  
Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates &gt;85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3830-3830
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Research Funding ◽  
Thymidine Incorporation ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1470-1470
Author(s):  
Jueqiong Wang ◽  
Chung Hoow Kok ◽  
Richard J. D'Andrea ◽  
Timothy P. Hughes ◽  
Deborah L. White
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Vehicle Control ◽  
Peroxisome Proliferator ◽  
Advisory Committees ◽  
Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

scholarly journals A New Therapeutic Hypothesis: Nonsense-Mediated Decay Is an Exploitable Target in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1937-1937
Author(s):  
Alexander Leeksma ◽  
Ingrid A.M. Derks ◽  
Brett Garrick ◽  
Torsten Trowe ◽  
Aldo Jongejan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Protein Production ◽  
Splicing Factors ◽  
Advisory Committees ◽  
Nonsense Mediated Decay

Abstract Background Nonsense-mediated decay (NMD) is a cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) as well as ~10% of normal mRNAs (Kurosaki and Maquat, 2016). NMD thus prevents translation of misfolded proteins, and potential activation of the unfolded protein response (UPR). Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 and ZRSR2 found in hematological as well as solid tumors, can lead to generation of aberrant mRNAs that contain PTCs. Aberrant splicing patterns in cancer cells can possibly result in increased pressure on the NMD machinery. CC-115, a potent inhibitor of mTOR kinase (TORK) and of DNA-dependent protein kinase, (DNA-PK; Mortensen et al., 2015; Tsuji et al., 2017), is in clinical development for the treatment of solid and hematologic malignancies (Thijssen et al., 2016). Preclinical data revealed an additional target of CC-115 and its differential effect on NMD. Our hypothesis was that a subset of tumor cells, especially hematologic tumors with high protein production and/or splicing factor mutations, would be susceptible to NMD inhibition by CC-115. Methods In total, 141 cell lines were screened for sensitivity to CC-115-mediated inhibition of proliferation and induction of cell death, in comparison to specific inhibition of TORK (CC-223). Isogenic DNA-PK knockout cell lines HCT116/HCT116 DNA-PK-/- and M059K/M059J DNA-PK-/- were treated with CC-115 and CC-223. Activity on NMD in vivo was tested using HCT-116 xenograft tumors treated with Vehicle or CC-115. Dependence on CC-115 sensitivity was determined using CRISPR/Cas9 technology of apoptosis or UPR genes in various MM cell lines. RNA sequencing was used for identification of potential targets in sensitive and resistant cell lines. Results A subset of cancer cell lines underwent cell death at sub-micromolar concentrations of CC-115 due to inhibition of NMD, but this was independent of mutations in splicing factors such as SF3B1. We next focused on MM cells as these generally produce high levels of (immunoglobulin) proteins and are prone to ER stress, and therefore potentially susceptible to NMD inhibition. Indeed, treatment with CC-115 resulted in activation of the UPR independent of TORK and DNA-PK inhibition, and cell death in 11/12 MM cell lines. Activity of CC-115 correlated strongly with cell death by the known ER-stress inducer, thapsigargin. Cell death by CC-115 occurred by the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax-Bak. Analysis of RNA sequencing data is ongoing and has indicated potential targets dictating sensitivity to CC-115-mediated cell death. Conclusions We describe that hematologic tumors with high protein production are specifically sensitive to CC-115, a novel and clinically exploitable inhibitor of NMD. This might lead to application in malignancies that depend on NMD to avoid excessive protein stress, such as multiple myeloma. Disclosures Garrick: Celgene: Employment. Trowe:Celgene: Employment. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Eldering:Celgene: Research Funding. Filvaroff:Celgene: Employment.

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 &gt; 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p&lt; 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.

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