Inhibition Of Wee1 Enhances The Anti-Leukemic Effects Of Antimetabolites In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1281-1281
Author(s):  
James B. Ford ◽  
Susan Fosmire ◽  
Annemie van Linden ◽  
Dmitry Baturin ◽  
Christopher C. Porter
Keyword(s):  
Combination Therapy ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Single Agent ◽  
Parp Cleavage

Abstract While some patients with acute leukemia are cured, for many subsets of patients current therapeutic strategies are not adequate. Novel therapeutic approaches are needed for patients with higher risk leukemias, including T-ALL and AML. We and others identified Wee1 as a potential target in AML cells using RNAi screening. We have validated chemosensitization to cytarabine by genetic and pharmacologic inhibition of Wee1 in AML cell lines and primary patient samples ex vivo. A Wee1 inhibitor, MK1775, is in clinical development. We sought to further our findings with a wider range of conventional anti-leukemia agents, to determine whether the functionality of p53 influences chemosensitization, and to determine the tolerability and efficacy of MK1775 in combination with cytarabine in a mouse model of leukemia. We have found that MK1775 synergistically inhibits proliferation of the T-ALL Jurkat cell line with several antimetabolite chemotherapeutics including cytarabine, 6-thioguanine, and methotrexate. In contrast, MK1775 does not sensitize Jurkats to doxorubicin or etoposide, suggesting specific sensitization to antimetabolites. The addition of MK1775 enhances the antimetabolite induced apoptosis, as measured by Annexin V/7-AAD staining, and PARP cleavage measured by Western blotting. As expected, the addition of MK1775 enhances DNA damage induced by cytarabine as measured by γH2AX staining and flow cytometry, although preliminary data suggest that this is not the only mechanism of enhanced cell death, as a substantial proportion of cleaved PARP+ cells does not stain for γH2AX. In addition, we have found that AML cell lines with both wild-type and mutated TP53 are sensitive to chemosensitization by Wee1 inhibition. Furthermore, in isogenic models of p53 dysfunction, we have found that the functionality of p53 does not influence chemosensitization. Lastly, in an aggressive mouse model of AML, we observed enhanced disease control and survival in mice treated with MK1775 and ARA-C as compared to ARA-C alone. Hematotoxicity associated with treatment was related to the duration of combination therapy, but was tolerated well with intermittent dosing. Taken together, these data indicate that Wee1 inhibition may enhance the efficacy of several clinically relevant anti-leukemia agents, particularly the antimetabolites, but not topoisomerase inhibitors. Further, they suggest caution about the use of p53 mutation as a biomarker predictive of response to Wee1 inhibition. Moreover, we show that the addition of MK1775 to cytarabine is tolerable and more effective than cytarabine alone in vivo. Ongoing studies are aimed at better understanding the mechanism of combinatorial effect and to determine whether combination therapy is more efficacious than single agent therapy in xenograft models of leukemia. These data provide justification for early phase clinical trials of MK1775 in combination with antimetabolites in patients with high risk acute leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Combination of CUDC-907 and Gilteritinib Shows Promising Antileukemic Activity in Vitro and In Vivo in Preclinical Models of FLT3-ITD AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1262-1262
Author(s):  
Tristan Knight ◽  
Xinan Qiao ◽  
Jun Ma ◽  
Holly Edwards ◽  
Lisa Polin ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Mouse Model ◽  
Cell Lines ◽  
Vehicle Control ◽  
Antileukemic Activity ◽  
Oral Gavage ◽  
In Vivo Efficacy ◽  
Flt3 Expression

Introduction FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are found in approximately one quarter of acute myeloid leukemia (AML) cases. Its presence results in constitutive activation of the FLT3 receptor tyrosine kinase and its downstream growth/pro-survival pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT, and confers a poor prognosis. Gilteritinib is a selective inhibitor of FLT3 recently approved by the Food and Drug Administration for treatment of patients with relapsed/refractory AML and a FLT3 mutation. However, gilteritinib exposure induces upregulation of FLT3 - a mechanism of resistance. Previously, we showed that CUDC-907, a dual PI3K/histone deacetylase inhibitor, downregulates FLT3 expression (Li X, et al. Haematologica. 2019; epub ahead of print). We therefore hypothesized that combining CUDC-907 with gilteritinib would abrogate FLT3 upregulation and expression, resulting in synergistic antileukemic activities against FLT3-mutated AML. Methods FLT3-ITD AML cell lines and primary patient samples were treated with gilteritinib or CUDC-907, alone or in combination at clinically achievable concentrations, and subjected to annexin V/propidium iodide staining and flow cytometry analysis to quantify apoptosis. Protein levels of FLT3, Bcl-2 family proteins, and key components of the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways were examined using western blotting. The impact of the observed alterations upon apoptosis were confirmed via overexpression, knockdown, and targeted inhibitor experiments. Real-time RT-PCR was used to determine FLT3 transcript levels. The FLT3-ITD AML cell line MV4-11 was used to generate a xenograft mouse model to assess in vivo efficacy of the two agents. Results CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines in vitro and patient samples ex vivo, with combined therapy. CUDC-907 abolished gilteritinib-induced expression of FLT3 in both cell lines and primary patient samples. Gilteritinib treatment reduced p-AKT, p-S6, and p-STAT5 and increased p-ERK, while CUDC-907 reduced p-AKT and p-ERK, and upregulated p-STAT5. The combination of gilteritinib and CUDC-907 decreased not only p-AKT and p-S6, but also p-ERK and p-STAT5. Targeted inhibition of ERK and JAK2/STAT5 signaling by SCH772984 and AZD1480, respectively, confirmed their roles in resistance to gilteritinib and CUDC-907 monotherapies, respectively. Combined gilteritinib and CUDC-907 treatment reduced expression of the anti-apoptotic BCL-2 family member Mcl-1 and increased expression of the pro-apoptotic protein Bim. MCL-1 overexpression and BIM knockdown partially rescued FLT3-ITD AML cells upon drug treatment, confirming their role in the antileukemic activity of combined gilteritinib and CUDC-907. To determine in vivo efficacy of the two agents, NSGS mice were injected with MV4-11 cells. Three days later, the mice were randomized into vehicle control (n=5), 40 mg/kg gilteritinib (oral gavage; n=5), 100 mg/kg CUDC-907 (oral gavage; n=5) or combination (40 mg/kg gilteritinib + 100 mg/kg CUDC-907; n=6) groups. CUDC-907 was given daily for 5 days on, 2 days off, for a total of 4 cycles. Gilteritinib was administered daily for 28 days. Both agents were well tolerated; maximal weight loss was 5.5%, 0.9%, and 6.7% in the CUDC-907, gilteritinib, and combination groups, respectively. Median survival of mice in the vehicle control group was 43 days. Median survival in the CUDC-907 monotherapy and gilteritinib monotherapy arm was 40.5 days and 104 days, respectively. One mouse in the combination therapy arm died on day 138, while the remaining 5 mice in the combination therapy arm continue to survive, as of time of writing (day 168), and are asymptomatic (Figure 1). Conclusion We confirmed that the combination of CUDC-907 plus gilteritinib synergistically induces apoptosis in both FLT3-ITD AML cell lines and primary patient samples, and that gilteritinib-induced FLT3 expression is abolished by CUDC-907. Cooperative inhibition of the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, as well as upregulation of Bim/downregulation of Mcl-1 all appear to contribute to this observed antileukemic synergy. Our cell line-derived xenograft mouse model provides strong evidence of in vivo efficacy and robust grounds for clinical translation of this therapeutic combination. Disclosures No relevant conflicts of interest to declare.

Efficacy of Panobinostat (LBH589) in Multiple Myeloma Cell Lines and In Vivo Mouse Model: Tumor-Specific Cytotoxicity and Protection of Bone Integrity in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1510-1510 ◽  
Author(s):  
Joseph D. Growney ◽  
Peter Atadja ◽  
Wenlin Shao ◽  
Youzhen Wang ◽  
Minying Pu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Bone Density ◽  
Mouse Model ◽  
Cortical Bone ◽  
Cell Lines ◽  
Single Agent ◽  
Synergistic Cytotoxicity

Abstract Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Here we report the effects of panobinostat on multiple myeloma (MM) cells in vitro and in a murine xenograft model in vivo. Panobinostat exhibited potent cytotoxic activity (IC50 <10 nM) against 8 MM cell lines (KMS-12PE, KMS-18, LP-1, NCI H929, KMS-11, RPMI8226, OPM-2, and U266). Panobinostat has been shown to affect signals involved in MM cell-cycle arrest and cell death, and to induce apoptosis via mitochondrial perturbation. In addition, panobinostat has been shown to selectively induce cell death of plasma cells isolated from MM patients without toxicity to normal lymphocytes or granulocytes. To investigate the effect of panobinostat in vivo, a disseminated luciferized MM.1S xenograft mouse model was treated with vehicle or panobinostat 15 mg/kg by intraperitoneal (i.p.) administration qd×5 for 3 weeks. Panobinostat treatment reduced the burden of MM.1S tumor cells to 22% treated over control (T/C) relative to vehicle-treated animals. In addition, MM.1S tumor-bearing mice treated with panobinostat displayed reduced trabecular and cortical bone damage relative to vehicle-treated animals. The mean ± SEM trabecular bone density and cortical bone density (% Bone Volume/Total Volume) of panobinostat-treated animals was 14.5% ± 2.0 and 98.1% ± 0.4, respectively, compared with 2.2% ± 0.3 and 89.1% ± 1.5 in vehicle-treated animals. In combination with the proteosome inhibitor bortezomib (BZ), panobinostat displayed significant synergistic cytotoxicity without additional toxicity to normal bone marrow stromal cells in vitro. In the MM.1S-luciferase tumor mouse model, combined treatment with panobinostat at 10 mg/kg i.p. qd×5 for 4 weeks and BZ at 0.2 mg/kg intravenously 1qw for 4 weeks reduced tumor burden to 7% T/C relative to vehicle, panobinostat alone (31% T/C), or BZ alone (44% T/C). Disease progression, measured as median time to endpoint (TTE) was improved from 37 to 54 days (P<0.05) by panobinostat and to 46 days by BZ (P<0.05). The combination treatment further improved clinical outcome relative to both single-agent treatment groups (P<0.05), extending the TTE to 73 days. In contrast to BZ, the immunomodulatory drug thalidomide (TH) had no significant single-agent activity at 150 mg/kg p.o. qd for 4 weeks. However, combination activity (18% T/C) was observed when TH was combined with a sub-efficacious dose of panobinostat (5 mg/kg, 64% T/C). Combination of panobinostat and TH increased the TTE to 50 days, compared with 37.5, 43, and 39.5 days (P<0.05), respectively, for the vehicle, panobinostat, or TH as single agents. These data demonstrate that panobinostat exhibits significant anti-proliferative and anti-tumor activities on MM cells both in vitro and in vivo. Panobinostat, as a single agent or in combination with BZ or TH, is a promising therapy for MM, and these studies may provide the rationale for clinical evaluation of panobinostat and BZ combination in the treatment of MM.

Anergy in CLL: Moving Towards the Clinic

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4677-4677
Author(s):  
Benedetta Apollonio ◽  
Tania Veliz Rodriguez ◽  
Cristina Scielzo ◽  
Maria Teresa Sabrina Bertilaccio ◽  
Lydia Scarfò ◽  
...  
Keyword(s):  
Mouse Model ◽  
B Cell ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Volume Growth ◽  
Lymphocytic Leukemia ◽  
Functional Features

Abstract B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease. CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors. In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p< 0,0001). To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy. Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth. Trametinb administration delayed tumour growth (p<0.05 starting at days 27) and inhibited leukemic cell dissemination in the peripheral blood, peritoneal cavity and bone marrow. In summary, our data further support the idea that blocking anergic pathways may be highly effective not only in vitro but also in vivo with potential clinical implications at least in the subset of patients whose cells are characterized by anergic features, including those with persistent lymphocytosis when treated with Ibrutinib. The preclinical efficacy shown by Trametinib, a drug already approved for clinical use, warrants the implementation of controlled studies in CLL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Combination Therapy of Bcl-2/X L dual Inhibitor AZD0466 with Acalabrutinib to Overcome Therapeutic Resistance in Aggressive R/R Mantle Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1867-1867
Author(s):  
Yijing Li ◽  
Yang Liu ◽  
Yuxuan Che ◽  
Joseph McIntosh ◽  
Alexa A Jordan ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Research Funding ◽  
Single Agent ◽  
Dual Inhibitor ◽  
Car T ◽  
Mantle Cell

Abstract Introduction As a rare form of non-Hodgkin's lymphoma, mantle cell lymphoma (MCL) is an aggressive subtype. This is largely due to frequent relapses after therapies including paradigm shifting therapies BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, and Bcl-2 inhibitor (Bcl-2i) venetoclax after long-term treatment in the clinic. Dysregulation of Bcl-2 and Bcl-X L, contributes to therapeutic resistance in MCL. AZD0466 is a novel and highly potent Bcl-2/X L dual inhibitor with active moiety AZD4320. Our preliminary data showed AZD4320 is potent in inhibiting cell viability of MCL cells (IC 50 = 1.6-78 nM). In this study, we assessed the combination efficacy of AZD4320/AZD0466 and acalabrutinib on preclinical MCL models. Methods Cell viability assay was performed to assess the in vitro efficacy of AZD4320 and acalabrutinib alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD4320 and acalabrutinib enhanced cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by immunoblotting. Since AZD4320 is limited in preclinical model due to physicochemical properties and dose limiting cardiovascular toxicity, AZD0466, the drug-dendrimer conjugate of AZD4320, was used for in vivo experiment. In vivo efficacy of AZD0466 (34 mg/kg, weekly, iv) and acalabrutinib (20 mg/kg, BID, oral) alone or in combination was evaluated using a Mino-venetoclax-R (Mino-R) cell xenograft model and a PDX model derived from an ibrutinib-CAR-T dual-resistant MCL patient. Results AZD4320 in combo with acalabrutinib inhibited cell proliferation synergistically in both ibrutinib/venetoclax-sensitive and -resistant cell lines (combination index = 0.17-0.93). Compared to vehicle or either single agent, the combination enhanced cell apoptosis by increasing pro-apoptotic markers cleaved caspase 3 and cleaved PARP. In the xenograft mouse model derived from venetoclax-resistant Mino-R cells, co-treatment of AZD0466 and acalabrutinib decreased tumor size significantly compared to vehicle (n = 5, p &lt; 0.0001) or either single agent (n = 5, p = 0.0118 and 0.0070, respectively). Furthermore, in the PDX mouse model derived from a patient relapsed subsequently from ibrutinib and CAR T therapy, the combination of AZD0466 and acalabrutinib inhibited tumor growth compared to vehicle or either single agent. Acalabrutinib or AZD0466 improved survival compared with vehicle by 14 days or 32 days, respectively. Compared to Acalabrutinib or AZD0466, the combination therapy extended survival by 25 days and 7 days, respectively. All mice tolerated the treatment dose without any weight loss compared to the vehicle or either single agent group. Conclusion Compared to AZD4320/AZD0466 and acalabrutinib, combination therapy demonstrated anti-MCL synergy both in vitro and in vivo. These findings suggest that targeting Bcl-2/X L and BTK is promising to overcome multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang: DTRM Biopharma (Cayman) Limited: Consultancy; BeiGene: Consultancy, Honoraria, Research Funding; Physicians Education Resources (PER): Honoraria; Anticancer Association: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Epizyme: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; BGICS: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria; Celgene: Research Funding; Genentech: Consultancy; Loxo Oncology: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Molecular Templates: Research Funding; Lilly: Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Oncternal: Consultancy, Research Funding; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Chinese Medical Association: Honoraria; Pharmacyclics: Consultancy, Research Funding; Juno: Consultancy, Research Funding; CStone: Consultancy; Bayer Healthcare: Consultancy; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Moffit Cancer Center: Honoraria; Mumbai Hematology Group: Honoraria.

Antimyeloma Efficacy of Plitidepsin (Aplidin®): From Bench to the Bedside.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1178-1178 ◽  
Author(s):  
Enrique M. Ocio ◽  
Constantine Mitsiades ◽  
M. Victoria Mateos ◽  
Patricia Maiso ◽  
Faustino Mollinedo ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Lines ◽  
Single Agent ◽  
In Vitro Studies ◽  
Parp Cleavage ◽  
Hematologic Toxicity ◽  
Cell Transplant ◽  
Significant Toxicity

Abstract Introduction Plitidepsin is a cyclic depsipeptide isolated from the marine tunicate, Aplidium albicans with promising antitumor activity. This work represents a comprehensive study (in vitro, in vivo and clinical) of its antimyeloma efficacy. Material & Methods In vitro studies were performed in 23 multiple myeloma (MM) cell lines and in cells from 16 MM patients. For the in vivo analysis a human plasmocytoma model in CB17-SCID mouse was used. Mice were randomized to receive Aplidin® 100 μg/Kg ip x 7 days/week (n=9), Aplidin® 140 μg/Kg ip x 5 days/week (n=7) or vehicle alone (n=9). The clinical efficacy of Aplidin® in relapsed/refractory patients was evaluated in a non-randomized two-stage Phase II, multicenter, clinical trial. Dosage of Aplidin® was 5 mg/m2 every 2 weeks. Results Aplidin® showed clear in vitro efficacy (IC50:1–10 nM) in the 23 cell lines tested including those resistant to dexamethasone, melphalan or doxorubicin. It was also active in the presence of microenvironment (IL-6, IGF-1 and BMSCs). Thirteen out of the 16 patient samples were sensitive to Aplidin® with >80% cell death in 8 cases and 60–80% in the remaining ones without significant toxicity in non tumor cells. Combination of Aplidin® with dexamethasone, bortezomib or lenalidomide showed clear potentiation. Aplidin® acts by inducing apoptosis with caspase−3, −7, −8, −9 and PARP cleavage. It also involves the activation of p38 and JNK signalling, Fas/CD95 translocation to lipid rafts and downregulation of Mcl-1 and myc. In mice studies, both schedules of treatment reduced tumor growth and increased survival with statistical differences in the group receiving 140 μg/Kg x 5d/week (p=0.04, Log Rank p=0.02). No significant toxicity was observed. These data provided the rationale for a clinical trial that has included 31 patients with relapsed/refractory MM. Median age was 65 years (47–82) and the median number of prior lines of therapy was 4 (range: 1–9) including autologous stem cell transplant (60%), thalidomide (58%) and bortezomib (48%). Out of the 26 evaluable patients, 2 (8%) achieved PR and 3 (12%) MR. Eight patients (31%) remained in stable disease (SD). Due to the synergism with dexamethasone observed in the in vitro studies, the protocol was amended to allow the addition of this agent in pts progressing after 3 cycles or with SD after 4 cycles. With a median follow-up of 14 months (range: 6.8–16.3), the time to progression in responding pts was 5.8 months (4.9–7.6). The most common G3-4 adverse events were fatigue (7%), serum creatine phosphokinase increase (7%), muscle toxicity (10%) and hepatic toxicity (10%). No significant hematologic toxicity or neuropathy was observed. Conclusion Aplidin® is effective both as a single agent and in combination with dexamethasone in the in vitro and in vivo settings. Its activity in relapsed/refractory MM patients is promising with an acceptable toxicity profile.

scholarly journals In Vivo Assessment of the Next Generation Microtubule-Destabilizing Agent AB8939 in Patient-Derived Xenograft Models of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5142-5142
Author(s):  
Armelle Goubard ◽  
Martine Humbert ◽  
Colin Mansfield ◽  
Olivier Hermine ◽  
Patrice Dubreuil ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Growth ◽  
Median Survival ◽  
Ex Vivo ◽  
Single Agent ◽  
Single Cycle ◽  
Advisory Committees ◽  
Proliferation Assays

AB8939 is a novel, synthesized, small-molecule microtubule-destabilizer drug with proven prolific and potent in vitro activity against numerous cancer cell lines. In vitro and ex vivo studies (reported separately) have determined that AB8939 is well-suited for the treatment of hematopoietic tumors, in particular relapsed/refractory or poor-prognosis acute myeloid leukemia (AML), notably being able to circumvent two major resistance mechanisms associated with AML (i.e. P-glycoprotein and myeloperoxidase-mediated resistance). The therapeutic potential of AB8939 was investigated further through a series of in vivo experiments using three patient derived xenograft (PDX) mouse models and a cytarabine (Ara-C) resistant mouse model (MOLM14). MOLM14 cells and selected PDX primary cells were transduced to constitutively express luciferase for bioluminescence monitoring of tumor growth. In an Ara-C-sensitive AML PDX mouse model (ex vivo IC50 response to Ara-C in survival/proliferation assays was 0.82 µM), AB8939 (6 mg/kg in weekly cycles of 5 consecutive days) showed a statistically significant, 10-fold decrease in the amount of blasts detected in blood following 14 days of treatment compared with control, and a superior treatment effect compared with Ara-C (single cycle of 10 mg/kg twice per day for 4 consecutive days) in terms of decreased blasts in blood. In an Ara-C-refractory AML PDX mouse model (ex vivo IC50 response to Ara-C in survival/proliferation assays was 6.4 µM), animals treated with single agent AB8939 (6 mg/kg in weekly cycles of 5 consecutive days) showed reduced disease progression compared with control and Ara-C (single cycle of 10 mg/kg twice per day for 4 consecutive days) as evidenced from at least 10-times fewer blasts in blood, spleen and bone marrow following 28 days of treatment. This effect was even more pronounced for the combination treatment of AB8939 and Ara-C, suggesting a synergistic response. In a PDX mouse model that is highly resistant to Ara-C (ex vivo IC50 response to Ara-C in survival/proliferation assays was 8.3 µM), AB8939 as a single agent or in combination with Ara-C showed a significant (P <0.001) decrease in tumor growth and reduction of blasts in blood with respect to Ara-C and control, following 27 days of treatment (8 animals per group). This improvement translated to survival benefit, with the single agent AB8939 cohort having a median survival of 89 days compared with 69 days and 65.5 days in the control and Ara-C cohorts, respectively. Indeed, all animals treated with single agent AB8939 were still alive at D83 post injection, which was 30 days after treatment was stopped. AB8939 as a single agent was well-tolerated with no toxicity-related deaths or impact on body weight. A greater treatment effect was again observed for the AB8939 plus Ara-C combination; however, clear signs of higher toxicity mean it will be imperative to optimize dosage of both AB8939 and Ara-C if used in combination. For the well-established xenografted MOLM14 mouse model, immune-deficient NSG (NOD scid gamma) mice (5 animals per group) were injected intravenously with MOLM14-luciferase cells and treated over a period of 21 days with single agent AB8939 (subcutaneous injection) at a dosage of 6 mg/kg every day or 12 mg/kg every other day; Ara-C (intraperitoneal injection, single cycle of 10 mg/kg twice per day for 4 consecutive days); or vehicle. AB8939 caused a significant dose-dependent reduction in tumor volume (p=0.001) and increased survival with respect to control or single agent Ara-C (median survival at 6 and 12 mg/kg was 39 and 42 days, respectively, corresponding to a 60% improvement compared with the control and Ara-C groups). A similar dosing schedule study showed single agent AB8939 at 6 mg/kg administered over 6 consecutive days (6 ON/1 OFF) was optimal with this cohort having a median survival of 59 days, corresponding to a 100% improvement over control. Overall, these in vivo data provide compelling proof-of-concept for AB8939 as a treatment of AML. AB8939 administered alone or in combination with Ara-C was demonstrated to significantly increase survival and reduce tumor growth as compared with single agent Ara-C in relevant animal models of AML. A first in human, phase 1 trial evaluating AB8939 in AML patients unfit to receive intensive chemotherapy in second and third-line has been initiated. Disclosures Goubard: AB Science: Employment. Humbert:AB Science: Employment. Mansfield:AB Science: Employment, Patents & Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

The Vk*MYC Mouse Model of Myeloma Identifies Successful Combination Therapies for the Treatment of Bortezomib Refractory Myeloma Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3015-3015 ◽  
Author(s):  
Marta Chesi ◽  
Stephen Palmer ◽  
Victoria Garbitt ◽  
P. Leif Bergsagel
Keyword(s):  
Clinical Trials ◽  
Mouse Model ◽  
Board Of Directors ◽  
Cell Lines ◽  
Human Disease ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Advisory Committees

Abstract Abstract 3015 Several preclinical models are available to assess the efficacy of novel anti-myeloma therapies, each of them with specific utilities. The most commonly utilized are: 1) Human myeloma cell lines (HMCLs) offer the advantage of being easily manipulated and well genetically characterized and therefore are best used to provide in-vitro target validation and to demonstrate specific target inhibition by a drug. On the other hand, they are highly proliferative and do not recapitulate the complexity of the human disease in an endogenous micro-environment. 2) Xenograft studies, in which HMCLs are injected into immunodeficient mice, serve the purpose of demonstrating that in-vivo target inhibition can be achieved under physiological conditions, but again fail to represent the indolence of human MM and its complex interaction with the BM stromal cells. Drug screenings performed on both these models tend inevitably to overestimate the antimyeloma activity of compounds that simply inhibit proliferation, but are ineffective in targeting the bulk of MM tumor. 3) Direct cytotoxic studies on primary patients cells, alone or in co-culture with stroma cell lines, are useful in assessing a pro-apoptotic activity in a way that is completely independent on proliferation. However, once again this in-vitro model does not capture the complete biology of human MM. The immuno-competent Vk*MYC mouse model of myeloma has already demonstrated high biological fidelity to the human disease, making it an ideal model to study the behavior of myeloma cells in the context of a native microenvironment and immune system. Furthermore, this model offers the advantage of studying both indolent, BM localized, untreated MM (primary MM), and, with the use of transplants into syngeneic hosts, advanced, more proliferative and refractory disease (secondary MM). Using the primary MM model, we have assessed the anti-myeloma activity of 30 known and novel compounds, many of which currently in clinical trials, and found that agents effective in the treatment of patients are active in this model, whereas agents ineffective in the treatment of patients are not. From this study, the most promising class of novel agents in clinical trials are the HDAC inhibitors vorinostat and panobinostat. Based on this promising single agent activity, we sought to model the effects of combination therapy in the secondary myeloma models that we specifically generated to be bortezomib refractory or multidrug resistance. Although transplanted mice treated with full dose single agent bortezomib, vorinostat or panobinostat showed no response and died within three weeks post transplant, recipient mice treated with bortezomib in combination with either vorinostat or panobinostat achieved complete response and are still alive 10 weeks post-transplant. We conclude that the bortezomib + HDAC inhibitors is an active combination that overcomes in vivo bortezomib resistance. Furthermore, the Vk*MYC model provides an excellent platform for the development of novel combinations aimed to the treatment of refractory MM disease. Disclosures: Bergsagel: Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Millennium: Speakers Bureau; Novartis: Speakers Bureau.

Anti-Vascular and Anti-Tumor Effects of the Vascular Disrupting Agent ASA404 (DMXAA) in Human Acute Leukemia Xenograft Models

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4293-4293
Author(s):  
Christopher W Schaefer ◽  
Deepika Lal ◽  
R Robert Vethanayagam ◽  
Joseph A Spernyak ◽  
Mukund Seshadri ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Vascular Function ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Vascular Disrupting Agent ◽  
Chemotherapy Drugs ◽  
Xenograft Models ◽  
Vascular Disrupting

Abstract Abstract 4293 Acute leukemia growth and expansion within the marrow microenvironment is linked to increased vascularization. Vascular delivery of chemotherapy drugs within the marrow space may also be an important mediator of anti-leukemic activity. We examined the effects of a tumor -vascular disrupting agent (tumor-VDA), ASA404 (DMXAA; Vadimezan), against human acute leukemia alone and in combination with anthracycline therapy. Exposure of human acute leukemia cells (HEL, Raji) to ASA404 (200–1000 mM) in vitro for up to 72 hours resulted in no evidence of direct cytotoxicity. However, contrast-enhanced magnetic resonance imaging (CE-MRI) performed in mice bearing subcutaneous HEL xenografts revealed an early increase in vascular permeability 4 hours after treatment with a single dose of ASA404 (25 mg/kg). This was followed by significant vascular disruption at 24 hours after therapy as determined by tumor immunohistochemistry. In systemic leukemia xenograft models established with luciferase-transfected HEL and Raji cells, continuous ASA404 treatment (20 mg/kg twice weekly) slowed overall systemic leukemia disease progression (as determined by whole animal bioluminescent imaging) in association with marked marrow vessel dilation, loss of vascular patency, and marked erythrocyte extravasation within the marrow microenvironment. These effects were accompanied by 20–30-fold elevations of circulating systemic TNF-alpha levels in ASA404 versus vehicle treated animals. Combining in vivo ASA404 treatment with anti-human VEGF antibody (bevacizumab) therapy further inhibited leukemia growth as compared with vehicle- or single-agent treated controls. However, concomitant administration of ASA404 with doxorubicin chemotherapy did not improve anti-leukemia effects and was in fact less effective than single agent therapy. In summary, our results demonstrate that ASA404 therapy is capable of mediating in vivo anti-tumor, anti-vascular and immunomodulatory effects in human acute leukemia models. Further strategies to modulate the marrow environment by combining a vascular disrupting agent with other biological agents for acute leukemia therapy may be warranted. However, given the time-dependent effects of VDAs such as ASA404 on tumor vascular function, combining VDAs with chemotherapy is likely to require further optimization of sequence and schedule of administration to ensure adequate marrow drug delivery and maximal therapeutic efficacy in hematological malignancy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

2021 ◽  
Author(s):  
Yu-bo Zhou ◽  
Yang-ming Zhang ◽  
Hong-hui Huang ◽  
Li-jing Shen ◽  
Xiao-feng Han ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Targets ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Preclinical Study ◽  
Parp Cleavage ◽  
Rpmi 8226 ◽  
Ongoing Phase

AbstractHDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

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