scholarly journals ONC212 Is a Potent Member of the Imipridone Class of Anti-Cancer Compounds That Induces p53-Independent Apoptosis in Hematological Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4059-4059
Author(s):  
Takenobu Nii ◽  
Jo Ishizawa ◽  
Ran Zhao ◽  
Jianfang Zeng ◽  
Dhruv Chachad ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Cell Lines ◽  
Research Funding ◽  
Time Course ◽  
Protective Factor ◽  
Hematological Malignancies ◽  
Human Tumor ◽  
Protein Translation ◽  
Clinical Activity ◽  
Anti Cancer

Abstract The functional or genetic inactivation of p53 hampers human tumor treatment. Therefore, novel therapeutic strategies are needed. ONC201 is a p53-independent inducer of apoptosis that is the founding member of the imipridone class of novel anti-cancer compounds, which possess a unique pharmacophore. We discovered that ONC201 exerts anti-tumor effects via ATF4 induction through activation of an atypical integrated stress response (ISR) (Ishizawa et al. and Kline et al, Sci Signal, 2016). Several clinical trials of ONC201 are ongoing in advanced cancers, showing a promising safety profiling and signs of clinical activity in both solid tumors and hematopoietic malignancies. In this study, we investigated the effects of ONC212, which has emerged as a highly potent member of the imipridone family, in preclinical models of hematological malignancies. ONC212 exerted potent and prominent apoptogenic effects on acute myeloid leukemia (AML) and mantle cell lymphoma (MCL) cell lines (e.g., ED50s of 141.0 nM in p53 wild-type OCI-AML3 cells, 105.7 nM in MOLM13 cells, and 265.2 nM in p53-null JeKo-1 cell lines). Time course analysis of apoptosis in OCI-AML3 cells showed that ONC212 takes more than 36 hours to start to induce apoptosis, which is similar to observations with ONC201. Next, we further examined similarities between ONC212 and ONC201 by evaluating the in vitro efficacy of ONC212 in ONC201-resistant (ONC201-R) cell lines that we have generated by chronic exposure of MCL and AML cell lines to ONC201, of which ED50s for ONC201 treatment at 72 hrs were all > 5 μM. Interestingly, the ONC201-R cell lines were more resistant to ONC212 than the isogenic ONC201-naïve cells (Figure 1), indicating that these cell lines are cross-resistant to ONC212. We previously proved that increased protein translation of the transcription factor ATF4 is one of the major molecular events involved in ONC201-induced apoptosis (Ishizawa et al., Sci Signal, 2016). Similarly, ATF4 protein abundance was increased by 24-hour treatment with ONC212. DDIT3 (CHOP) gene, a target of ATF4, was transcriptionally upregulated in parallel with its target genes GADD34, DR5 and TRIB3 in ONC212-treated JeKo-1 and OCI-AML3 cells by 24 hrs after treatment (Figure 2). Of note, ONC201 was reported to transcriptionally induce TRAIL in a p53-independent manner in solid tumors (Allen et al., Sci Transl Med, 2013), but it was not operational in hematological cell lines (Ishizawa et al., Sci Sig 2016). Consistently, we also confirmed that ONC212 does not increase TRAIL mRNA in MCL (JeKo-1) and AML (OCI-AML3) cells. BCL-2 is a protective factor for cells under endoplasmic reticulum stress, which is one way to activate ISR. Therefore, we investigated whether the BCL-2 inhibitor ABT-199 sensitizes hematopoietic malignant cells to ONC212. Apoptosis was significantly higher in the combination than either drug alone in MCL and AML cell lines even in THP-1 and OCI-AML3 cells that are relatively resistant to ONC201/212 and/or ABT-199 (Figure 3), suggesting that this combination could overcome the resistance to either of agents. Indeed, the combination was also synergistic in the OCI-AML3 ONC201-R cell line (Figure 3). Taken together, our preclinical studies suggest that ONC212 is a promising and potent new member of the impridone class of anti-cancer compounds that warrants further development in hematological malignancies. The combination of ONC212 with ABT-199 is attractive, considering that acquired resistance after a short-term response remains a clinical challenge with ABT-199. Disclosures Konopleva: AbbVie: Research Funding; Genentech: Research Funding. Allen:Oncoceutics Inc.: Employment. Stogniew:Oncoceutics Inc.: Employment, Equity Ownership. Andreeff:Oncoceutics Inc.: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Pharmacoinformatics and Preclinical Studies of NSC765690 and NSC765599, Potential STAT3/CDK2/4/6 Inhibitors with Antitumor Activities against NCI60 Human Tumor Cell Lines

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 92
Author(s):  
Bashir Lawal ◽  
Yen-Lin Liu ◽  
Ntlotlang Mokgautsi ◽  
Harshita Khedkar ◽  
Maryam Rachmawati Sumitra ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Tumor ◽  
Cancer Cell Lines ◽  
Tumor Cell Lines ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Anti Cancer

Signal transducer and activator of transcription 3 (STAT3) is a transcriptional regulator of a number of biological processes including cell differentiation, proliferation, survival, and angiogenesis, while cyclin-dependent kinases (CDKs) are a critical regulator of cell cycle progression. These proteins appear to play central roles in angiogenesis and cell survival and are widely implicated in tumor progression. In this study, we used the well-characterized US National Cancer Institute 60 (NCI60) human tumor cell lines to screen the in vitro anti-cancer activities of our novel small molecule derivatives (NSC765690 and NSC765599) of salicylanilide. Furthermore, we used the DTP-COMPARE algorithm and in silico drug target prediction to identify the potential molecular targets, and finally, we used molecular docking to assess the interaction between the compounds and prominent potential targets. We found that NSC765690 and NSC765599 exhibited an anti-proliferative effect against the 60 panels of NCI human cancer cell lines, and dose-dependent cytotoxic preference for NSCLC, melanoma, renal, and breast cancer cell lines. Protein–ligand interactions studies revealed that NSC765690 and NSC765599 were favored ligands for STAT3/CDK2/4/6. Moreover, cyclization of the salicylanilide core scaffold of NSC765690 mediated its higher anti-cancer activities and had greater potential to interact with STAT3/CDK2/4/6 than did NSC765599 with an open-ring structure. NSC765690 and NSC765599 met the required safety and criteria of a good drug candidate, and are thus worthy of further in-vitro and in-vivo investigations in tumor-bearing mice to assess their full therapeutic efficacy.

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

The Design and Synthesis of Novel Phenothiazine Derivatives as Potential Cytotoxic Agents

2019 ◽  
Vol 17 (1) ◽  
pp. 57-67
Author(s):  
Yepeng Luan ◽  
Jinyi Liu ◽  
Jianjun Gao ◽  
Jinhua Wang
Keyword(s):  
Cell Lines ◽  
High Efficiency ◽  
Human Cancer ◽  
Human Tumor ◽  
Cytotoxic Agents ◽  
Cancer Drug ◽  
Human Cancer Cell Lines ◽  
Incidence And Mortality ◽  
Anti Cancer

Background: Cancer incidence and mortality have been increasing and cancer is still the leading cause of death all over the world. Despite the enormous progress in cancer treatment, many patients died of ineffective chemotherapy and drug resistance. Therefore, the design and development of anti-cancer drugs with high efficiency and low toxicity is still one of the most challenging tasks. Tricyclic heterocycles, such as phenothiazine, are always important sources of scaffolds for anti-cancer drug discovery. Methods: In this work, ten new urea-containing derivatives of phenothiazine coupled with different kinds of amine motifs at the endpoint through a three carbon long spacer were designed and synthesized. The structures of the synthesized compounds were elucidated and confirmed by 1H NMR and HRMS. All the synthesized compounds were tested for their antitumor activity in vitro against the proliferation of PC-3 cells, and the compounds with best potency entered further cytotoxicity evaluations against other 22 human tumor cell lines. Mechanism was also studied. Results: From all data, it showed that among all 10 target compounds, TTi-2 showed the best effect in inhibiting the proliferation of 23 human cancer cell lines while TTi-2 without obvious inhibitory effect on normal cell. Furthermore, our results also showed that TTi-2 could inhibit migration, invasion and colony formation of MDA-MB-231 cells. Finally, TTi-2 can induce arrest of cell cycle at G0/G1 phase and cell apoptosis by activating the caspase 3 activity. Conclusion: All these results suggested that TTi-2 might be used as a promising lead compound for anticancer drug development.

Activity of Plk Inhibitor BI2536 on Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2764-2764
Author(s):  
Douglas W. McMillin ◽  
Joseph Negri ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Jana Jakubikova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Solid Tumors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Human Tumor ◽  
Cyclin B1 ◽  
Myeloma Cell ◽  
Ic50 Values ◽  
Presence And Absence

Abstract Context: Novel therapeutic strategies targeting cell cycle regulation are attractive for multiple myeloma (MM) because of the increased proliferative index in advanced drug-resistant disease. Having previously studied the role of Cdk inhibition in MM, we have looked at the cell cycle-related polo kinases (PLKs), because their expression is associated with adverse prognosis in solid tumors. We report preclinical studies on the anti-MM activity of the PLK1/2/3 small molecule inhibitor BI2536. Methods/Results: We tested 39 human tumor cell lines by MTT colormetric assay, including MM (n=26), T-ALL (n=8), solid tumors (n=5), as well normal human tissues. BI2536 exhibited activity in the low nano-molar range with IC50 values <10 nM for the most sensitive cells lines, which included several MM lines. BI2536 exhibited minimal activity against normal PBMCs, unstimulated or PHA-stimulated, with IC50 values greater than the highest dose tested (i.e. 80 nM). Myeloma cell lines were further tested in the presence and absence of exogenous IL-6 (10ng/mL) and IGF-1 (50ng/mL) and exhibited the ability to overcome the cytokine-induced resistance observed with other anti-MM agents (e.g. Dex or Doxo). Interestingly, several stromal responsive myeloma cell lines, including MM.1S, MM.1R, H929 and INA-6 were more sensitive to BI2536 in the presence of HS-5 stromal cells compared to the stromal unresponsive cell line OPM2, which was equally sensitive in the presence and absence of stromal cells. In addition, myeloma cell lines co-cultured with osteoclasts (OC) exhibited comparable activity in the presence and absence of OCs. Cell cycle analysis showed that treatment with BI2536 causes rapid G2/M arrest and increased G0/G1 phase events in KMS18 cells. Mechanistic studies revealed that Akt, MAPK, cyclin B1, cyclin D1 and cdk1 levels decrease in response to BI2536 treatment, while caspase-3 and PARP are cleaved within 8 hrs of drug treatment at 20 nM. Interestingly, Notch and phospho-histone H3 levels increased in response to treatment. Gene expression profiling analysis further validated the finding that BI2536 functions distinctly from other anti-MM agents, since there was not an effect on transcriptional signatures of proteasome, NF-kB or IRF4 activity following BI2536 treatment in KMS18 cells. In addition, higher doses of BI2536 preferentially killed side-population cells (SP cells) compared to the main population (MP), as shown by Hoechst staining. Importantly, immunohistochemisty revealed that MM.1S cells treated with BI2536 were unable to recruit alpha-tubulin to mitotic centrosomes and form bipolar spindles, which is compatible with the role of polo kinases in mitotic spindle formation. We also evaluated a series of combinations of this agent with conventional (e.g. dexamethasone, doxorubicin) and novel (e.g. bortezomib) anti-MM agents. No evidence of antagonism with any of these anti-MM agents was observed, indicating that combinations of BI2536 may be feasible in clinical settings with current anti-myeloma regimens. Conclusion: Proteins pivotal for cell cycle progression represent promising targets for treating highly proliferating tumors. Treatment of MM with a PLK inhibitor provides evidence that polo kinases are promising targets for MM therapy. Importantly, BI2536 activity was enhanced in the presence of stromal cells, providing evidence that this class of compounds will be active in the tumor microenvironment.

Preclinical Characterization of PWT143, a Novel Selective and Potent Phosphatidylinositol 3-kinase Delta (PI3K delta) Inhibitor with Ex-Vivo Activity in Hematologic Malignancies.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2907-2907 ◽  
Author(s):  
Marie O'Farrell ◽  
Richard Ventura ◽  
Albert Tai ◽  
Jeffrey W Tyner ◽  
Marc M Loriaux ◽  
...  
Keyword(s):  
Lymph Node ◽  
Tumor Cells ◽  
Mechanism Of Action ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Clinical Activity ◽  
Direct Inhibition ◽  
Ic50 Values ◽  
Proliferation Assays ◽  
Pi3k Delta

Abstract Abstract 2907 Early clinical trials with small molecule inhibitors that target kinases in the B Cell Receptor (BCR) signaling pathway have demonstrated promising activity in B cell malignancies. These kinases include PI3K delta, Bruton's tyrosine Kinase (BTK) and spleen tyrosine kinase (SYK), with clinical validation demonstrated by the inhibitors GS-1101 (CAL-101), ibrutinib, and fostamatinib respectively. The clinical observations with GS-1101 and ibrutinib include rapid lymph node shrinkage and high lymph node response rate in refractory CLL as well clinical activity in indolent NHL and MCL, and in DLBCL for ibrutinib. In addition to B cells, PI3K delta is expressed in other hematopoietic cells such as T cells, mast cells and neutrophils, and plays a role in cellular signals transmitted by immunoreceptors such as FcεR, FcγR, and chemokine receptors. Therefore, inhibitors of PI3K delta may have utility in diverse hematological malignancies, in addition to those in B cells. PWT143 is a highly selective PI3K delta inhibitor that has been selected as a development candidate. PWT143 exhibits low nanomolar potency in cellular phosphorylation assays against PI3K delta. PWT143 exhibits cellular selectivity of 2200-, 30- and 700-fold against the alpha, beta and gamma isoforms, with no activity against approximately 500 other kinases tested, including mTOR. Low nM potency has also been demonstrated in a whole blood functional assay of basophil activity which is encouraging for translation to the clinic. The activity of PWT143 has been profiled in survival and proliferation assays in a panel of 12 human hematological malignancy cell lines, and compared to GS-1101, ibrutinib, and fostamatinib. The cell lines included DLBCL, Burkitts lymphoma, and lymphoblastic leukemias. PWT143 IC50s ranged from 30 nM to 4 μM with the majority approximating 1μM, while the comparator molecules exhibited higher IC50values: 250 nM to > 10 μM for GS-1101, 250 nM to 9.5 μM for ibrutinib, and 400 nM to > 10 μM for fostamatinib. Viability/proliferation assays were also performed in peripheral blood cells freshly isolated from patients with various hematological malignancies. In CLL samples, PWT143 displayed IC50 values < 100 nM in 3 of 4 cases, in marked contrast to GS-1101 or ibrutinib which exhibited IC50 values >10 μM for the majority of these samples tested. Potent activity was also observed for PWT143 in primary AML samples with IC50s in the 100 nM range or lower for 3 of 5 cases tested, but generally > 1 μM for GS-1101 or Ibrutinib. In previously frozen CLL and AML patient samples procured from commercial sources, PWT143 similarly exhibited several-fold lower IC50values than GS-1101 or ibrutinib. These data suggest increased sensitivity of CLL and AML patient samples to PWT143. The lack of activity of GS-1101 and ibrutinib at low micromolar concentrations in the primary cell assays is consistent with the published mechanism of action. Rather than a direct inhibition of tumor cell viability, the major axis of clinical activity is inhibition of stromal-tumor interactions mediated by BCR “inside-out” signaling which normally maintains tumor cells in the lymph node. Accordingly, the clinical activity of GS-1101, ibrutinib and fostamatinib is associated with marked lymphocytosis due to release of tumor cells from the lymph nodes into peripheral blood, observed in the initial weeks of treatment and often persists for many months. The direct inhibition of viability by PWT143, as well as the established stromal-mediated mechanism of action of PI3K delta inhibitors, may translate to increased clinical activity for PWT143. PWT143 is a potent and selective PI3K delta inhibitor, and preclinical data indicate that it is an attractive candidate for clinical development. Disclosures: O'Farrell: Pathway Therapeutics: Employment, own equity as a Pathway employee Other. Ventura:Pathway Therapeutics: Employment, own equity as a Pathway employee Other. Tai:Pathway Therapeutics: Consultancy. Tyner:Pathway Therapeutics: Research Funding. Loriaux:Pathway Therapeutics: Research Funding. Mahadevan:Pathway Therapeutics: Research Funding. Morales:Pathway Therapeutics: Research Funding. Brown:Pathway Therapeutics: Consultancy. Matthews:Pathway Therapeutics: Employment, Own equity as a Pathway employee Other.

Mitochondrial Complex I Inhibition with Iacs-010759 in T-ALL Preclinical Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4028-4028 ◽  
Author(s):  
Natalia Baran ◽  
Jennifer Molina ◽  
Antonio Cavazos ◽  
Karine Harutyunyan ◽  
Ningping Feng ◽  
...  
Keyword(s):  
Cell Lines ◽  
Complex I ◽  
Research Funding ◽  
Stress Test ◽  
Metabolic Reprogramming ◽  
Survival Duration ◽  
Cellular Respiration ◽  
Clinical Activity ◽  
Viable Cell Number ◽  
Sensitivity Pattern

Abstract Metabolic reprogramming of the key energy-generating pathways has been long recognized as one of the key oncogenic properties of cancer including leukemia. While accelerated glycolysis is considered to be most common feature of tumors, reliance on oxidative phosphorylation (Oxphos) as a major energy source has been reported for various tumor types. IACS-010759 is a novel OxPhos inhibitor(OxPhosi) that blocks cellular respiration through inhibition of complex I (Molina et al., AACR2016 Abstract #335) and considered as validated drug with clinical relevance in AML and solid tumors. Treatment of adult T-ALL remains unsatisfactory, with approximately one-third of patients experience disease relapse, and novel treatment strategies are warranted. In this study, we report pre-clinical activity of IACS-010759 in T-ALL models and characterize a cellular metabolic profile of T-ALL. Analysis of a panel of T-ALL cell lines showed that IACS-010759 significantly reduced viability measured by CTG assay in all cell lines tested (Notch mutant: Pf382, 1301, Jurkat, MOLT-4, P12-Ichikawa and Notch wt: T-ALL1). T-ALL cells displayed high sensitivity pattern to OxPhos inhibition with EC50 between 0,001 and 10 nM at day 5 analyzed by CTG assay (Fig.1). This reduction of cell viability was primarily due to cell cycle arrest demonstrated by reduction in EdU uptake, and moderate induction of apoptosis in selected T-ALL cell lines. In primary T-ALL samples from patients with newly diagnosed or relapsed/refractory ALL (n=2), in vitro 5-day treatment with IACS-010759 reduced viable cell number at EC50 of 13 nM and 45 nM, respectively. In primary human T-ALL PDX xenografts study, daily oral administration of IACS-010759 at 7.5mg/kg/qd was well tolerated, caused significantly reduced circulating leukemia burden and extended median survival duration (Fig.2). The mitochondrial fuel usage that characterizes Oxphos dependency in T-ALL cell line PF382 was analyzed by Mito fuel Test using the Seahorse Bioscience XF96 Analyzer. Among all three energy sources, PF382 depends most on free fatty acids (FA), indicating strong coupling to Oxphos and TCA cycle (Fig.3). Treatment of T-ALL with IACS-010759 had effectively inhibited FA-stimulated mitochondrial respiration indicated by decreased oxygen consumption rates (OCR) (Fig.4A). However, the cells maintain an ability to generate energy via glycolysis, indicated by high extracellular acidification rate (ECAR) in both, control and IACS-treated groups (Fig.4B). Next, mitochondrial function of T-ALL cells (PF382, Jurkat, 1301, P12Ischikawa, MOLT4, TALL1) was investigated using Mito Stress Test in Seahorse Bioscience XF96 Analyzer. IACS-010759 exposure for 2 hrs caused a striking dose-dependent decrease in basal and maximal OCR, reduction of proton leak and ATP production (Fig.5A, B, C), confirmed by the decreased ATP/ADP and NADH/NAD ratios measured by luminescence assays (ADP/ATP Glow assay, NADH/NAD Glow assay), consistent with inhibition of Oxphos. Conclusions: Taken together, these data provide information about metabolic profiling of T-ALL and indicate that Oxphos inhibition constitutes a novel therapeutic approach that targets a unique metabolic vulnerability of T-ALL cells. Further preclinical evaluation of Oxphos inhibitors in T-ALL is warranted. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Preliminary evidence of activity of pralatrexate in relapsed/refractory ovarian cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e15557-e15557
Author(s):  
Nashat Y. Gabrail ◽  
Carrie L. Smith
Keyword(s):  
Ovarian Cancer ◽  
Renal Impairment ◽  
Renal Insufficiency ◽  
Solid Tumors ◽  
Hematological Malignancies ◽  
Folate Receptor ◽  
Ca 125 ◽  
Clinical Activity ◽  
Weekly Dose ◽  
The Impact

e15557 Background: Pralatrexate, a novel antifolate, is approved in the US for the treatment of relapsed/refractory peripheral T-cell lymphoma at a dose of 30mg/m2 weekly for 6 weeks of a 7-week cycle. Previous studies in solid tumors have focused on a q2w schedule at 190mg/m2. As pralatrexate is 30-40% renally excreted, an ongoing renal impairment study is investigating the impact of mild, moderate and severe renal impairment on pralatrexate distribution in patients with hematological malignancies and solid tumors. As folate receptor alpha is frequently overexpressed in epithelial ovarian cancers, we hypothesized that ovarian cancer may be a target for pralatrexate treatment and therefore have included a number of ovarian cancer patients in this trial. Methods: Patients with solid tumors and hematological malignancies were enrolled in a phase I clinical trial. Patients are treated in cohorts depending on renal function normal, mild renal insufficiency, moderate renal insufficiency and severe non-dialysis dependent renal failure. This analysis is limited to 6 patients with refractory ovarian cancer. The dose of pralatrexate is 30mg/m2 weekly for 6 weeks of a 7-week cycle, with dose adjustment depending on toxicity. Results: The 6 patients, all with stage 4 refractory ovarian cancer had received 4 or more prior chemotherapy regimens and had refractory disease, defined as, nonresponsive to the last chemotherapy regimen or progression within 3 months of the last dose of chemotherapy. Of the 6 patients, 2 experienced objective responses lasting 9 months and 4 months respectively. Disease response was assessed by CA- 125 and RECIST criteria. Two patients experienced stable disease for 4 months and 5 months respectively. One patient had grade 5 mucositis after the second weekly dose and one patient had progressive disease after disease stabilization for 3 months. Conclusions: Pralatrexate given at a dose of 30mg/m2 to patients with refractory ovarian cancer has demonstrable activity that needs to be confirmed in well-designed efficacy trials. Importantly, this is the first demonstration of clinical activity in a solid tumor at the currently approved dose.

scholarly journals Targeting Replicative Stress to Treat Hematological Disorders

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2419-2419
Author(s):  
Francesca Cottini ◽  
Giovanni Tonon ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
Kenneth C Anderson
Keyword(s):  
Dna Damage ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Advisory Committees ◽  
Hematological Disorders ◽  
Replicative Stress ◽  
Hematological Cancers ◽  
Equity Ownership

Abstract Background: Combinations of chromosomal translocations, copy-number variations, somatic mutations, and clonal heterogeneity that characterize hematological cancers make every patient unique from a genetic point of view. This variety creates a true challenge for tailored therapy. We have previously described that myeloma (MM) cells present signs of ongoing DNA damage, and activate an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis. Here we further characterize the mechanisms of DNA damage and replicative stress in MM, and we extend this knowledge to other hematological malignancies to evaluate a novel and possibly shared approach to synthetic lethality (1). Results: We studied a panel of MM cell lines together with acute myeloid, lymphoid leukemia and lymphoma cell lines. Several cell lines have demonstrable ongoing DNA damage, activate ATR and CHK1 and also present with signs of replicative stress, such as 53BP1, RPA and RAD51 foci. We next evaluated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients, comparing them with normal plasma cells. Specifically, we identified a subset of patients, representing around 20 percent of individuals with MM that show this signature and also present with an unfavorable prognosis due to a more aggressive disease. Interestingly, in a multivariate analysis, this signature was independent from other poor prognostic criteria, including proliferation and the presence of MMSET/FGFR3 or MAF translocations (2), hence representing a potential novel prognostic signature (3). Gene-set enrichment studies are ongoing in other hematological disorders. However, preliminary data show that subsets of patients with other hematological malignancies also present with intense over expression of genes belonging to the instability signature when compared to normal B cells, in a very similar fashion to that seen with MM. An intact ATR/CHK1 pathway is crucial for the survival of tumor cells in vivo, especially in the presence of activated oncogenes. For instance, Em-myc transgenic mice develop B-cell lymphomas with intense replicative stress that can be blocked by crossing the Em-myc transgenic mice with a hypomorphic Atr mouse strain (Atr-Seckel; Atr S/S). We therefore decided to exploit the concept of replicative stress overload, impeding the capacity of the cells for repairing the excess of damaged DNA. We then silenced ATR using shRNAs, the upstream protein involved in the control of stalled replication origins, in two MM cell lines (H929 and OPM-2) and in the Jurkat cell line. Critically, inhibition of ATR caused a reduction in cellular growth and induction of apoptosis. A similar phenotype was observed using VE-821, a specific ATR inhibitor. Finally, a broad panel of MM cells and leukemia cell lines was used to confirm these growth inhibitory effects. Conclusion: Replicative stress is present in multiple groups of patients with aggressive types of MM or leukemia. Strategies which couple pre-existing high rates of DNA damage with reduced DNA repair can specifically cause apoptosis of malignant cells and encouragingly spare normal ones, thus providing a strong rationale for potential clinical benefit to those cohorts of patients with otherwise very unfavorable outcomes. (1) Cottini F, Hideshima T, Xu C, Sattler M, Dori M, Agnelli L, et al. Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers. Nature medicine. 2014;20(6):599-606. (2) Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S, et al. The molecular classification of multiple myeloma. Blood. 2006;108(6):2020-8. (3) Cottini F, Teru Hideshima, Rikio Suzuki, Yu-Tzu Tai, et al. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma. Cancer Discovery ahead of print. Disclosures Richardson: Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene Corporation: Consultancy; Oncocorp: Equity Ownership; acetylon pharmaceuticals: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy.

scholarly journals Targeting BCL-XL By Protac DT2216 Effectively Eliminates Leukemia Cells in T-ALL Pre-Clinical Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3870-3870
Author(s):  
Qi Zhang ◽  
Sajid Khan ◽  
Xuan Zhang ◽  
Vinitha Mary Kuruvilla ◽  
Sanaz Ghotbaldini ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Research Funding ◽  
Patent Application ◽  
Medical Sciences ◽  
Cytochrome C Release ◽  
Anti Cancer ◽  
Equity Ownership ◽  
Dose Dependent ◽  
Pdx Models

T-ALL is an aggressive hematologic malignancy arising from immature T-cell precursors. Previous studies identified dependence of T-ALL (with a notable exception of early T-cell precursor (ETP) ALL) on BCL-XL (Chonghaile, Cancer Discovery 2014; Khaw, Blood 2016). However, BCL-XL specific inhibitors exhibit on-target toxicity of thrombocytopenia, restricting the use in acute leukemias (Vogler, Blood 2011). DT2216, a novel BCL-XL specific proteolysis targeting chimera (PROTAC), targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL ubiquitination and degradation selectively in cells express VHL (Khan, ASH 2018). Platelets lack VHL expression and therefore are spared from destruction by DT2216. Here we studied the pre-clinical efficacy of DT2216 in T-ALL cell lines in vitroand in vivousing T-ALL patient-derived xenograft (PDX) models. We first analyzed anti-apoptotic proteins (BCL-XL, BCL-2, MCL-1) expression in 4 B-ALL (LAZX2, MUTZ5, RS4:11, BALL1) and 6 T-ALL cell lines (SUPT1, KOPT1, Loucy, CCRF-CEM, PF384, Jurkat) by immunoblotting. This analysis demonstrated that ALL cell lines generally co-express BCL-XL and BCL-2 (Figure 1A). To identify functional dependencies, we utilized BH3 profiling that measures cytochrome C release after priming cells with BH3 peptides selectively targeting pro-survival BCL-2 family proteins in 4 B-ALL and 3 T-ALL cell lines. Similarly, cells were co-dependent on several anti-apoptotic members as shown by higher cytochrome c release in response to BIM, BID and BMF peptides targeting multiple anti-apoptotic proteins, and lower response to FS-1, ABT-199, HRK, MS-1, targeting individual anti-apoptotic members (Figure 1B). Analysis of the 3 B-ALL and 3 T-ALL PDX lines identified similar patterns that ALL cells are co-dependent on several anti-apoptotic members. Notably, we observed high cytochrome C release in response to mBAD that targets BCL-2 and BCL-XL; in addition, two of the three T-ALL PDXs, but none B-ALL PDX, responded to BCL-XL specific peptide HRK and to DT2216 confirming a functional role of BCL-XL in T-ALL survival. Next, we studied the sensitivity of ALL cells to ABT-199, DT2216 and the combination, in comparison with dual BCL-2/BCL-XL inhibitor ABT-263. DT2216 treatment (24hrs) caused a dose-dependent reduction of cellular viability in all 6 T -ALL and 3 B-ALL lines (except for BALL1 with complex karyotype refractory to all agents) measured by Cell TiterGlo assay, with T-ALL cells demonstrating a log higher sensitivity compared to B-ALL. In contrast, 5 out of 6 T-ALL lines (all besides ETP line Loucy) had no response to ABT-199, while 3 B-ALL lines showed dose-dependent response. All lines except BALL1 responded to ABT-263 (Figure 1C). Notably, the combination of DT2216 with ABT-199 synergistically reduced cell viability, with average CI of 0.3 (range 0.1-0.7 in all lines besides BALL1) (Figure 1D). Immunoblotting of DT2216 treated cells confirmed dose-dependent, on-target BCL-XL degradation as early as 6 hrs (Figure 1E). We next tested the therapeutic efficacy of DT2216 alone or combined with chemotherapy in T-ALL PDX models. NSG mice were engrafted with T-ALL PDX CU76 and D115. After documenting bone marrow (BM) engraftment by flow cytometry in BM aspirates on Day 14 post cell injection, mice were randomized to receive vehicle, chemotherapy ("VDL", VCR 0.15mg/kg, Dexa 5mg/kg, L-ASP 1000U/kg, ip., qw), DT2216 (15mg/kg, ip., q4d) or their combination for 3 weeks. Mice tolerated DT2216 therapy well, with no platelet toxicity by whole blood count 24hrs post the first and last DT2216 dosing. DT2216 reduced leukemia burden, delayed leukemia progression (Fig 1G) and significantly extended mice survival in both models. VDL chemotherapy had no effect on ALL progression in CU76 model and showed efficacy similar to DT2216 in D115 model; of importance, VDL+ DT2216 combination resulted in significant extension of survival in both chemoresistant and chemosensitive models (Figure 1F). In summary, T-ALL cells are functionally dependent on BCL-XL for survival and are highly sensitive to DT2216, while B-ALL are largely BCL-2 dependent and respond to BCL-2 inhibitors such as ABT-199. DT2216 alone and in particular when combined with chemotherapy reduced leukemia burden and prolonged survival in T-ALL PDX models. This study suggests targeting BCL-XL by DT2216 represents highly effective and safe adjunct therapeutic modality in T-ALL. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Zhang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Ghotbaldini:CPRIT Research Grant: Research Funding. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents. Konopleva:Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

scholarly journals Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3800-3800 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Zhihong Fang ◽  
Courtney DiNardo ◽  
Elias Jabbour ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Leukemia Cell ◽  
Clinical Activity ◽  
Guanine Nucleotide ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis

Abstract Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with <100 µM FF-10501-01. Decreased intracellular concentrations of guanine nucleotides were observed in MOLM13 and SKM1 cell lines treated with 3 to 30 µM of FF-10501-01 for 24 hours. Proliferation was partially rescued after 72 hours of treatment with 3 µM guanosine and FF-10501-01 in MOLM-13, HL60 cells and their HMA-resistant derivatives. No treatment synergy was observed with the combination of FF-10501-01 with HMAs in MOLM-14 and HL-60 or their HMA-resistant cell lines. In summary, FF-10501-01 produced potent anti-proliferative and apoptotic effects on AML cell lines through inhibition of de novo guanine nucleotide synthesis. In view of these pre-clinical findings, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ³ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. Sixteen patients (12 AML, 4 MDS) have been enrolled in 5 dose cohorts (50 - 400 mg/m2 PO BID) for 14 days on/14 days off each 28-day cycle, including 8 M and 8 F. Median (range) values: age 75.3 yrs (59.1 - 88.6); bone marrow blasts for AML patients 40.5% (12 - 71), for MDS patients 10% (6 - 13), or 30% overall (6 - 71); and prior treatment regimens 2.5 (1 - 6). All patients relapsed from, or progressed on, prior HMAs. Mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A and/or MDM2 were present in 4/16 (25%) patients. The median number of FF-10501-01 cycles received to date is 1.5 (range 1 - 10). No DLTs or drug-related serious adverse events (AEs) have been observed and FF-10501-01 has been very well tolerated through 5 - 10 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related Gr 4 prolonged thrombocytopenia and Gr 4 prolonged neutropenia were reported in one patient at 200 mg/m2 BID. Two partial responses (PRs) have been achieved in 1 patient each at 50 and 100 mg/m2 BID after 3 cycles, 7 (50%) patients demonstrated long-term stable disease over 2 - 10 cycles, and 4 patients have remained on study drug through 5 - 10 cycles and are still ongoing. Updated safety and efficacy data, including PK/PD, will be presented at the meeting. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS. Preclinical activity was seen in multiple leukemia cell lines. In a Phase 1 trial, clinical activity with PRs, prolonged disease stabilization and a highly tolerable safety profile were observed. The Phase 2 expansion phase will be initiated soon. Disclosures DiNardo: Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Smith:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutic, Inc.: Employment. Madden:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

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