In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1496-1496 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Andrea Ghelli Luserna Di Rorà ◽  
Maria Vittoria Verga Falzacappa ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Single Agent ◽  
Time Dependent ◽  
Leukemia Cells ◽  
In Vivo Models

Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

scholarly journals PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii4-iii4
Author(s):  
A Bruning-Richardson ◽  
H Sanganee ◽  
S Barry ◽  
D Tams ◽  
T Brend ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Combination Treatment ◽  
Single Agent ◽  
Drug Penetration ◽  
In Vivo Models ◽  
Human Astrocytes ◽  
Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

scholarly journals The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Vera Miranda-Gonçalves ◽  
João Lobo ◽  
Catarina Guimarães-Teixeira ◽  
Daniela Barros-Silva ◽  
Rita Guimarães ◽  
...  
Keyword(s):  
Dna Damage ◽  
Germ Cell ◽  
Differential Expression ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Germ Cell Tumors ◽  
Migration And Invasion ◽  
In Vivo Models

Abstract Background Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors. Methods Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo. Results We demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11. Conclusions VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

scholarly journals 47 Preclinical studies of dianhydrogalactitol (VAL-083) in DIPG, as single agent or as a combination with AZD1775 or radiation

2018 ◽  
Vol 45 (S3) ◽  
pp. S9-S10
Author(s):  
Xiaodong Yang ◽  
Anne Steino ◽  
Jeffrey Bacha ◽  
Dennis Brown ◽  
Sabine Mueller
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Cell Cycle Progression ◽  
Single Agent ◽  
Chemotherapeutic Agents ◽  
Phase Cell ◽  
Dna Double Strand Breaks

Despite decades of trials, the prognosis for diffuse intrinsic pontine gliomas (DIPG) remains dismal. DIPG is inoperable and standard treatment is radiation alone, as the addition of chemotherapeutic agents, such as temozolomide, have not improved survival. In addition to inherent chemoresistance, treatment of DIPG is impeded by an intact blood-brain barrier (BBB). VAL-083 is a structurally unique bi-functional DNA-targeting agent that readily crosses the BBB. VAL-083 forms interstrand DNA crosslinks at N7-guanine, resulting in DNA double-strand breaks (DSB), S/G2-phase cell-cycle arrest, and ultimately cancer cell death. We have previously demonstrated that VAL-083 is able to overcome temozolomide-resistance in vitro and in vivo, and that its cytotoxicity is independent of the DNA-repair enzyme O6-methylguanine DNA-methyltransferase (MGMT). MGMT is almost universally expressed in DIPG and its expression is strongly correlated with temozolomide-resistance. VAL-083’s distinct mechanism-of-action suggests the potential for combination with inhibitors of DNA DSB repair or S/G2 cell-cycle progression (e.g. Wee1 inhibitor AZD1775). Here, we investigated the effects of VAL-083 in combination with radiation, AZD1775 or irinotecan (topoisomerase inhibitor) in three DIPG cell-lines: SF10693 (H3.1), SF8628 (H3.3) and NEM157 (H3.3). VAL-083 showed activity at low uM-concentration in all three cell-lines. In addition, VAL-083 showed synergy with AZD1775 in all three cell-lines. Combined with its ability to cross the BBB, accumulate in brain tumor tissue and overcome MGMT-related chemoresistance, these results suggest VAL-083 as a potentially attractive treatment option for DIPG as single agent or in combination with AZD1775. Combination studies with radiation are ongoing and will be presented at the meeting.

scholarly journals Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1945-1945
Author(s):  
Kylee H Maclachlan ◽  
Andrew Cuddihy ◽  
Nadine Hein ◽  
Carleen Cullinane ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Response ◽  
Single Agent ◽  
Resistant Cell ◽  
Rdna Transcription

Abstract Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4080-4080
Author(s):  
Yuhuan Zheng ◽  
Jing Yang ◽  
Liang Zhang ◽  
Jianfei Qian ◽  
Jairo Matthews ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Compound A

Abstract Abstract 4080 Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Efficacy and mechanism of the combination of PARP and CDK4/6 inhibitors in the treatment of triple-negative breast cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Xiuzhi Zhu ◽  
Li Chen ◽  
Binhao Huang ◽  
Xiaoguang Li ◽  
Liu Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Damage ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Single Agent ◽  
Wnt Signalling ◽  
Tnbc Cell

Abstract Background PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients with BRCA mutations, and their efficacy is even more limited in triple-negative breast cancer (TNBC) due to clinical primary and acquired resistance. Here, we found that the efficacy of the PARPi olaparib in TNBC can be improved by combination with the CDK4/6 inhibitor (CDK4/6i) palbociclib. Methods We screened primary olaparib-sensitive and olaparib-resistant cell lines from existing BRCAmut/TNBC cell lines and generated cells with acquired olaparib resistance by gradually increasing the concentration. The effects of the PARPi olaparib and the CDK4/6i palbociclib on BRCAmut/TNBC cell lines were examined in both sensitive and resistant cells in vitro and in vivo. Pathway and gene alterations were assessed mechanistically and pharmacologically. Results We demonstrated for the first time that the combination of olaparib and palbociclib has synergistic effects against BRCAmut/TNBC both in vitro and in vivo. In olaparib-sensitive MDA-MB-436 cells, the single agent olaparib significantly inhibited cell viability and affected cell growth due to severe DNA damage. In olaparib-resistant HCC1937 and SUM149 cells, single-agent olaparib was ineffective due to potential homologous recombination (HR) repair, and the combination of olaparib and palbociclib greatly inhibited HR during the G2 phase, increased DNA damage and inhibited tumour growth. Inadequate DNA damage caused by olaparib activated the Wnt signalling pathway and upregulated MYC. Further experiments indicated that the overexpression of β-catenin, especially its hyperphosphorylation at the Ser675 site, activated the Wnt signalling pathway and mediated olaparib resistance, which could be strongly inhibited by combined treatment with palbociclib. Conclusions Our data provide a rationale for clinical evaluation of the therapeutic synergy of the PARPi olaparib and CDK4/6i palbociclib in BRCAmut/TNBCs with high Wnt signalling activation and high MYC expression that do not respond to PARPi monotherapy.

scholarly journals DIPG-53. CHARACTERIZING THE ROLE OF PPM1D MUTATIONS IN THE PATHOGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGS)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii297-iii297
Author(s):  
Prasidda Khadka ◽  
Zachary Reitman ◽  
Sophie Lu ◽  
Graham Buchan ◽  
Rachel Hartley ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Radiation Treatment ◽  
Ectopic Expression ◽  
Loss Of Function ◽  
In Vivo Models ◽  
Cycle Arrest ◽  
Diffuse Intrinsic Pontine Gliomas

Abstract INTRODUCTION We have previously found that up to 15% of all DIPGs harbor mutations in PPM1D, resulting in the expression of an activated and truncated PPM1D (PPM1Dtr). Here we evaluate the mechanisms through which PPM1Dtr enhances glioma formation and identify its associated therapeutic vulnerabilities. METHODS We have developed multiple in vitro and in vivo models of PPM1D-mutant DIPGs and applied quantitative proteomic and functional genomic approaches to identify pathways altered by PPM1Dtr and associated dependencies. RESULTS PPM1D mutations are clonal events that are anti-correlated to TP53 mutations. We find ectopic expression of PPM1Dtr to be sufficient to enhance glioma formation and to be necessary in PPM1D-mutant DIPG cells. In addition, endogenous truncation of PPM1D is sufficient to enhance glioma formation in the presence of mutant H3F3A and PDGFRA. PPM1Dtr overexpression attenuates g-H2AX formation and suppresses apoptosis and cell-cycle arrest in response to radiation treatment. Deep scale phosphoproteomics analyses reveal DNA-damage and cell cycle pathways to be most significantly associated with PPM1Dtr. Furthermore, preliminary analysis of genome-wide loss-of-function CRISPR/Cas9 screens in isogenic GFP and PPM1Dtr overexpressing mouse neural stem cells reveal differential dependency on DNA-damage response genes in the PPM1Dtr overexpressing cells. Consistent with PPM1D’s role in stabilizing MDM2, PPM1D-mutant DIPG models are sensitive to a panel of MDM2 inhibitors (Nutlin-3a, RG7388, and AMG232). CONCLUSION Our study shows that PPM1Dtr is both an oncogene and a dependency in PPM1D- mutant DIPG, and there are novel therapeutic vulnerabilities associated with PPM1D that may be exploited.

scholarly journals Efficacy and Mechanism of the Combination of PARP and CDK4/6 Inhibitors in the Treatment of Triple-negative Breast Cancer

2020 ◽  
Author(s):  
XIUZHI ZHU ◽  
LI CHEN ◽  
Binhao Huang ◽  
Xiaoguang Li ◽  
Yang Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Damage ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Triple Negative ◽  
Single Agent ◽  
Wnt Signaling Pathway ◽  
Tnbc Cell

Abstract Background: PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients with BRCA mutations and their efficacy is even more limited in triple-negative breast cancer (TNBC) due to clinical primary and acquired resistance. Here, we found that the efficacy of PARPi in TNBC can be improved with CDK4/6 inhibitors (CDK4/6i).Methods: We screened primary PARPi-sensitive and resistant cell lines from existing BRCAmut/TNBC cell lines and generated cells with acquired PARPi resistance by gradually increasing the concentration. The effects of the PARPi olaparib and the CDK4/6i palbociclib on BRCAmut/TNBC cell lines were examined in both sensitive and resistant cells in vitro and in vivo. Pathway and gene alterations were assessed mechanistically and pharmacologically.Results: We demonstrated for the first time that the combination of PARPi and CDK4/6i has synergistic effects against BRCAmut/TNBC both in vitro and in vivo. In the PARPi-sensitive MB436 cells, the single agent olaparib significantly inhibited cell viability and affected cell growth due to severe DNA damage. In the PARPi-resistant HCC1937 and SUM149 cells, single-agent olaparib was ineffective due to potential homologous recombination (HR) repair, and the combination of PARPi and CDK4/6i greatly inhibited HR during the G2 phase, increased DNA damage and inhibited tumor growth. Inadequate DNA damage caused by PARPi activated the Wnt signaling pathway and upregulated MYC. Further experiments indicated that the overexpression of β-catenin, especially its hyperphosphorylation at the Ser675 site activated the Wnt signaling pathway and mediated PARPi resistance, which could be strongly inhibited by the combined treatment with CDK4/6i.Conclusions: Our data provide a rationale for the clinical evaluation of the therapeutic synergy of PARPi and CDK4/6i in BRCAmut/TNBCs with high Wnt signaling activation, high MYC expression and do not respond to PARPi monotherapy.

Filanesib (ARRY-520) Demonstrates Potent and Rapid Activity in Preclinical Models of MM, Dependent on Bcl-2 Family Expression, and Synergistic with Dexamethasone and IMiDs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4710-4710
Author(s):  
Susana Hernández-García ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Teresa Paíno ◽  
Ana Alicia López-Iglesias ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Cyclin B1 ◽  
Mitotic Checkpoint ◽  
Triple Combination ◽  
Apoptotic Protein ◽  
Apoptotic Proteins

Abstract Introduction: Multiple myeloma (MM) is characterized by the presence of complex karyotypes and chromosome instability, suggesting that cell cycle checkpoints are defective. Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle proteins (KSP), which are required to establish mitotic spindle bipolarity, driving centrosome separation. Filanesib monotherapy has demonstrated clinical activity in heavily pretreated MM patients (Lonial et al, ASH 2013). In this work we aimed to explore the preclinical activity of filanesib alone and in combination with IMiDs. Methods: In vitro activity of filanesib alone and in combination with IMiDs (thalidomide, lenalidomide and pomalidomide) was evaluated in MM cell lines by MTT assay and Annexin V, Propidium Iodide and DiOC6 analysis by flow cytometry, Western Blot and immunofluorescence. Synergy was quantified with combination indices (CI) by Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice. Results: Filanesib demonstrated significant activity in a broad panel of 11 MM cell lines, with 48-hour IC50 values ranging between 0.3 and 5 nM. Interestingly, the highest activity was observed in drug-resistant cell lines such as OPM-2 and RPMI-LR5. We next evaluated whether the cell death MoA was dependent on apoptosis or blockade of proliferation. Time response experiments performed in three different cell lines with different sensitivity to filanesib (OPM-2, MM1S and U266) showed accumulation of cells in G2/M, followed by loss of the mitochondrial membrane potential and activation of apoptosis. Accordingly, Western blot analysis demonstrated an activation of the mitotic checkpoint indicated by an increase in Cyclin B1, and activation of apoptosis with PARP, and caspase-3 and -7 cleavage. Furthermore, filanesib activity was very rapid as 15 minutes of exposure was sufficient to exert all of the apoptotic and cell cycle effects observed at 48 hours. Immunofluorescence microscopy using alpha-tubulin demonstrated that filanesib induces monopolar spindle formation. The sensitivity of MM to filanesib has been previously correlated with the cell-dependency of the anti-apoptotic protein Mcl-1. We, therefore, studied the basal levels of six Bcl-2 family members (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the 11 cell lines and observed a correlation between the basal levels of these proteins and drug sensitivity. In particular, we confirmed the relationship with the anti-apoptotic proteins Mcl-1 and Bcl-2 and demonstrated that conversely to what is observed with proteasome inhibitors, cells with high basal levels of pro-apoptotic Bax or Bak were more resistant to filanesib. Moreover, treatment with filanesib induced a clear decrease of Mcl-1 in the 3 cell lines analyzed (U266, MM1S and OPM-2) that coincided with a decrease of Bcl-2 in the most sensitive cell line, OPM-2. Finally, we evaluated the activity of filanesib in combination with IMiDs and dexamethasone. In vitro studies showed a synergistic effect of filanesib with dexamethasone (CI: 0.21), and with all IMiDs, being most pronounced with pomalidomide (CI: 0.09). Of note, this triple combination demonstrated the highest synergistic activity (CI: 0.06). These results were confirmed in vivo where the triple combination of filanesib, dexamethasone, and pomalidomide was also synergistic, with a significant reduction of tumor growth of up to 50 days, which correlated with a statistically significant survival improvement. Mechanistic studies on the combination are ongoing. Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. It was identified that this activity is dependent on the anti-apoptotic protein Mcl-1 and pro-apoptotic proteins Bax and Bak. Furthermore, in animal xenograft studies, filanesib exhibited robust synergism in combination with dexamethasone and any IMiD, pomalidomide being the most synergistic. These data are the rationale for the clinical trial "Pomdefil" which uses this combination in patients with relapsed refractory MM. The trial will begin soon as a collaboration of the Spanish Myeloma Group (GEM). This work was supported in part by Array BioPharma. Disclosures Humphries: Array Biopharma: Employment. Tunquist:Array Biopharma: Employment. Mateos:Array Biopharma: Honoraria. Ocio:Array Biopharma: Honoraria, Research Funding.

The Hybrid Molecule, Edo-S101, Impairs Double Strand Breaks Repair in Multiple Myeloma and Synergizes with Bortezomib and Dexamethasone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5354-5354 ◽  
Author(s):  
Ana Alicia López-Iglesias ◽  
Ana Belen Herrero ◽  
Laura San-Segundo ◽  
Susana Hernández-García ◽  
Lorena González-Méndez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Comet Assay ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Ex Vivo ◽  
Hybrid Molecule ◽  
Cycle Arrest

Abstract Introduction. EDO-S101 is a hybrid molecule of bendamustine plus vorinostat, new in its class. Our group has previously demonstrated that EDO-S101 is effective in vitro in MM cell lines independently of p53 state, and also in a murine plasmacytoma model where it decreases tumor growth and prolongs survival with respect to bendamustine and/or vorinostat treatment. The objective of this work was to gain further insights into the efficacy of EDO-S101, its mechanism of action and its combination with other drugs used in MM. Methods. The mechanism of action was assessed by western blot, comet assay, immunohistochemistry, and flow cytometry. Homologous recombination (HR) efficiency was calculated using chromosomally integrated green fluorescent protein reporter construct-based assay. The efficacy of different combinations was studied in vitro (HMCLs), in vivo (murine plasmacytoma model CB-17 SCID mice) and ex vivo (cells from patients). Results. In addition to the activity of EDO-S101 in MM cell lines we demonstrated that it was active ex vivo in cells isolated from 7 MM patients, with median IC50 of 5 µM (ranging from 1,8 to 8 µM), some of them previously exposed and resistant to alkylators such as melphalan. Interestingly, EDO-S101 could also overcome alkylators-resistance in vitro, as it was active in melphalan resistant cells (U266-LR7 and RPMI8226-LR5). EDO-S01 was also effective in the presence of factors that confer proliferative advantage to plasma cells, like IL-6, IGF or co-culture with mesenchimal cells hMSC-TERT. Regarding its mechanism of action, we found that the apoptosis induced by EDO-S101 was caspase-independent but calpain-dependent, since PD150606, an inhibitor of this protein could overcome EDO-S101-induced apoptosis, whereas the caspase inhibitor Z-VAD -FMK did not. This data was consistent with the finding that under treatment with EDO-S101, MM1S cells showed AIF (apoptotic inducing factor) translocation from the mitochondria into the nucleus. Interestingly, the release of this pro-apoptotic protein from the mitochondria could be mediated by calpains, as it has been described in literature. We subsequently demonstrated that EDO-S101 causes DNA damage, as revealed by the phosphorylation and subsequent activation of several components of the DNA Damage Response (DDR) such as ATM, H2AX, chk1, chk2 or p53, and the induction of DNA fragmentation, that was detected by the comet assay. EDO-S101 was also found to induce cell cycle arrest in different phases depending on the dose and cell line. It has previously been suggested that DACi may impair DNA repair by inhibiting homologous recombination (HR), a pathway related with genomic instability and progression, very active in MM. Therefore we next evaluated the efficiency of HR using a reported construct that was chromosomally integrated in two MM cell lines, JJN3 and U266. Treatment with EDO-S101 significantly reduced the efficiency of HR in both cell lines, by 50% and 20% of untreated controls respectively. Finally, we tested potential combinations with other antimyeloma agents like lenalidomide and thalidomide; and also with proteasome inhibitors (bortezomib, carfilzomib and oprozomib). EDO-S101 potentiated the activity of all these agents, but the most synergistic combination was that including Bortezomib + Dexamethasone (CI 0,4). This combination was also evaluated in vivo, where it significantly decreased tumor growth and prolonged survival compared to agents in monotherapy and in double combinations. We are currently deepening into the mechanism of action of this combination. Conclusions. EDO S101 is active ex vivo in cells isolated from patients and is able to overcome resistance to alkylators. It induces caspase-independent apoptosis, and cell cycle arrest in MM cell lines. These effects are due to the potent DNA damage which is enhanced by HR impairment induced by the hybrid molecule. Moreover, the combination with bortezomib and dexamethasone is especially attractive to be taken into the clinical setting. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy.

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