scholarly journals Identification and validation of a non-genetically encoded vulnerability to XPO1 inhibition in malignant rhabdoid tumors – expanding patient-driven discovery beyond the N-of-1

2021 ◽  
Author(s):  
Lianna J. Marks ◽  
Daniel Diolaiti ◽  
Prabhjot Mundi ◽  
Ervin S. Gaviria ◽  
Allison R. Rainey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Export ◽  
Multimodality Treatment ◽  
Protein Activity ◽  
Pediatric Solid Tumors ◽  
Patient Derived Xenograft ◽  
Novel Approach ◽  
Rhabdoid Tumors

AbstractMalignant rhabdoid tumors (MRTs) are rare, aggressive pediatric solid tumors, characterized by a 22q11 deletion that inactivates the SMARCB1 gene. Outcomes remain poor despite multimodality treatment. MRTs are among the most genomically stable cancers and lack therapeutically targetable genetic mutations. We utilized metaVIPER, an extension of the Virtual Inference of Protein-activity by Enriched Regulon (VIPER) algorithm, to computationally infer activated druggable proteins in the tumor of an eight month old patient and then expanded the analysis to TCGA and TARGET cohorts. In vitro studies were performed on a panel of MRT and atypical teratoid/rhabdoid tumor cell lines. Two patient-derived xenograft (PDX) mouse models of MRT were used for in vivo efficacy studies. MetaVIPER analysis from the patient’s tumor identified significantly high inferred activity of nuclear export protein Exportin-1 (XPO1). Expanded metaVIPER analysis of TCGA and TARGET cohorts revealed consistent elevations in XPO1 inferred activity in MRTs compared to other cancer types. All MRT cell lines demonstrated baseline activation of XPO1. MRT cell lines demonstrated in vitro sensitivity to the XPO1 inhibitor, selinexor which led to cell cycle arrest and induction of apoptosis. Targeted inhibition of XPO1 in patient-derived xenograft models of MRT using selinexor resulted in abrogation of tumor growth. Selinexor demonstrates efficacy in preclinical models of MRT. These results support investigation of selinexor in a phase II study in children with MRT and illustrate the importance of an N-of-1 approach in driving discovery beyond the single patient.Statement of Translational RelevanceWe describe the patient-driven discovery of XPO1 activation as a non-genetically encoded vulnerability in MRTs. The application of metaVIPER analysis to tumors lacking actionable oncogenic alterations represents a novel approach for identifying potential therapeutic targets and biomarkers of response. Our preclinical validation of selinexor confirms XPO1 inhibition as a promising therapeutic strategy for the treatment of MRT.

scholarly journals Proteasome inhibition as a therapeutic approach in atypical teratoid/rhabdoid tumors

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Andrew Morin ◽  
Caroline Soane ◽  
Angela Pierce ◽  
Bridget Sanford ◽  
Kenneth L Jones ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
High Grade Glioma ◽  
Proteasome Inhibition ◽  
Atypical Teratoid ◽  
Rhabdoid Tumors ◽  
Approved Drugs

Abstract Background Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%–45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. Methods We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. Results BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood–brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. Conclusions MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.

A phase I trial of MK 2206 in children with refractory solid tumors: A Children’s Oncology Group study.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9581-9581
Author(s):  
Christine L. Phillips ◽  
Maryam Fouladi ◽  
John Peter Perentesis ◽  
Sarah Leary ◽  
Renee M. McGovern ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phase I ◽  
Solid Tumors ◽  
Phase I Trial ◽  
Pediatric Solid Tumors ◽  
Biological Studies ◽  
Grade 3 ◽  
Rhabdoid Tumors

9581 Background: AKT, a serine threonine protein kinase, is activated downstream of phosphatidylinositol 3-kinase (PI3K) which transmits signals from cytokines, growth factors and oncoproteins to multiple targets. Activated AKT regulates survival, proliferation, and growth. The PI3K/AKT pathway is downstream of most of the common growth factor tyrosine kinase receptors in cancer, e.g., EGFR, HER2, IGFR, etc., and is a driver of tumor progression in many cancers. AKT protein kinase is activated in many pediatric solid tumors, including glioblastoma, malignant rhabdoid tumors, neuroblastoma, synovial sarcoma, rhabdomyosarcoma and medulloblastoma. MK2206, an oral allosteric AKT1, 2,3 inhibitor, has demonstrated antitumor activity in in vitro and in vivo cancer models.A phase I trial evaluating MK2206 was conducted in children with refractory solid tumors. Methods: Using a rolling-6 design, MK2206 was administered either once every 7 days (schedule 1), or once every other day (schedule 2) in a 28-day cycle. Serial PK studies were obtained on day 1, cycle 1 and trough samples were obtained on days 7, 14, 21 and 28. Biological studies included analysis of PI3K/PTEN/AKT-cell signaling pathway in pre and post-therapy in PBMC and in tumors at diagnosis or recurrence. Results: Forty-five patients [23 males, median age 13.6 years (range 3.1-21.9)] with malignant glioma (14), ependymoma (4), hepatocellular carcinoma (3), gliomatosis cereberi (2) or other tumors (22) were enrolled; 34 were fully evaluable for toxicity (schedule 1 n=17; schedule 2 n=17). Schedule 1 DLTs included: grade 3 dehydration in 1/6 patients at 28 mg/m2; grade 4 hyperglycemia and neutropenia in 1/6 patients at 45 mg/m2. There were no DLTs at 35 mg/m2 and dose level 4 (58 mg/m2) is currently open for patient accrual with one enrollment. Schedule 2 DLTs included: grade 3 alkaline phosphatase in 1/6 patients at 90 mg/m2; grade 3 rash in 1/6 patients at 120 mg/m2), and grade 3 rash in 2/6 patients at 155 mg/m2. Conclusions: The recommended pediatric phase II dose of weekly MK2206 is 120 mg/m2 and the last cohort of patients to the every other day dosing schedule of MMK206 is enrolling. PK and PD data are currently being analyzed and will be presented.

scholarly journals The Effects of PI3K-δ/γ Inhibitor, Duvelisib, in Mantle Cell Lymphoma in Vitro and in Patient-Derived Xenograft Studies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3016-3016 ◽  
Author(s):  
Jack Wang ◽  
Victoria Zhang ◽  
Taylor Bell ◽  
Yang Liu ◽  
Hui Guo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Oral Gavage ◽  
Patient Derived Xenograft ◽  
Pdx Model ◽  
Mantle Cell

Abstract Background: Mantle cell lymphoma (MCL) is an incurable subtype of B-cell lymphoma. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was approved by the FDA for the treatment of MCL in patients previously treated. In our prior multicenter Phase 2 clinical trial, the overall response rate in relapsed/refractory MCL was 68%, with a median progression-free survival (PFS) of 13.9 months. However, the majority of MCL patients treated with ibrutinib relapsed; in these relapsed patients, the one-year survival rate was only 22%. Therefore, there exists an urgent need for additional novel targeted therapies to improve the mortality rate in these patients. In this study, we assessed the in vitro and in vivo effects of duvelisib, a PI3K-δ,-γ inhibitor, in MCL. Methods: The PI3K/AKT/mTOR and other cell survival signaling pathways were investigated by RNASeq and reverse phase protein array (RPPA) in ibrutinib-sensitive and -resistant MCL samples. The expression of PI3K isoforms, α, β, γ, and δ was tested in 11 MCL cell lines, patient and patient-derived xenograft (PDX) MCL cells by western blot analysis. We then investigated the growth inhibition and apoptosis of duvelisib (IPI-145, Infinity Pharmaceuticals, Inc.) in MCL cells by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and Annexin V-binding assay (BD Biosciences). We established a primary MCL-bearing PDX model and passaged the primary MCL tumor to next generations. Mice were administrated with 50 mg/kg duvelisib daily by oral gavage. Tumor burden and survival time were investigated in the MCL-PDX model. Results: We found that the PI3K/AKT/mTOR signaling pathway was activated in both primary and acquired ibrutinib-resistant MCL cell lines and PDX MCL cells. We immunoblotted PI3K isoforms, α, β, γ, and δ in 11 MCL cell lines and the result demonstrated that both ibrutinib-sensitive and ibrutinib-resistant MCL cells dominantly expressed PI3K-δ and -γ. Next, we tested the effects of duvelisib on these MCL cells. Duvelisib had effects on the growth inhibition and apoptosis in both ibrutinib-sensitive and ibrutinib-resistant MCL cells as good as the PI3K-δ inhibitor, idelalisib (Cal-101, GS-1101). The PI3K-δ isoform could play a very important role in PI3K-mediated signals in MCL. We then investigated the effects of duvelisib in vivo through our established MCL-bearing PDX mouse models. These models are created by inoculating the primary tumor cells from MCL patients into a human fetal bone chip implanted into NSG mice to provide a microenvironment that reconstitutes the human environment. MCL tumor mass was then passaged to next generations for therapeutic investigation of duvelisib. Mice were treated with 50 mg/kg duvelisib daily by oral gavage. Our data demonstrated that duvelisib significantly inhibited tumor growth and prolonged survival of MCL-PDX mice. Conclusion: Duvelisib, an oral dual inhibitor of PI3K-δ,-γ, inhibits MCL growth both in vitro and in PDX mice. These preclinical results suggests duvelisib may be effective in the treatment of patients with relapsed/refractory MCL. Disclosures No relevant conflicts of interest to declare.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 237-237 ◽  
Author(s):  
Michael P. Rettig ◽  
Matthew Holt ◽  
Julie Prior ◽  
Sharon Shacham ◽  
Michael Kauffman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Employment Equity ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

Discovery and validation of the nuclear export protein CRM1 (XPO1) as a new target for the treatment of renal cell carcinoma (RCC).

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 411-411
Author(s):  
Hiromi Inoue Wettersten ◽  
Michael Kauffman ◽  
Sharon Shacham ◽  
Yosef Landesman ◽  
Joy Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Export ◽  
Human Kidney ◽  
In Vivo Studies ◽  
Tumor Growth Inhibition ◽  
Phase I Clinical Trials ◽  
New Therapeutic Approaches ◽  
Xenograft Mice

411 Background: The currently available targeted therapies for RCC have had limited success, so it is imperative to discover new therapeutic approaches for metastatic RCC. In a search for new such targets, we have identified inhibitors of CRM1 (XPO1, exportin1), the major karyopherin that mediates the export of most tumor suppressor proteins from the nucleus. To expand our initial work on the first generation of CRM1 inhibitors, we now evaluate KPT-330, an orally available Selective Inhibitor of Nuclear Export (SINE), the best tolerated among a series of CRM1 inhibitors that is currently in phase I clinical trials. Methods: The RCC cell lines (Caki-1 and 786-O) and a primary normal human kidney (NHK) cell line were treated with KPT-330, and MTT assays were performed. The cells were subjected to immunofluorescence and immunoblotting for appropriate proteins. Caki-1 xenograft mice were treated with KPT-330 for 15 days, and tumor volume was assessed. Results: KPT-330 selectively attenuated CRM1 levels and caused dose-dependent toxicity (EC50 < 1 µM) through apoptosis in all RCC cells. In untreated RCC cells, p21 was localized in the nucleus, where it likely functions as a cell cycle arrest protein, and in the cytosol, where it functions as an anti-apoptotic protein. However, in NHK cells, p21 was confined to the nucleus. KPT-330 increased p53 and p21 and confined them to the nucleus in both NHK and RCC cells. KPT-330 given orally inhibited RCC growth in xenograft mice (85.3 % inhibition, p < 0.001). Moreover, KPT-330 showed synergism with a Bcl-2 inhibitor ABT-737 in vitro, indicating the potential for combination therapy with a CRM1 inhibitor and Bcl-2 inhibitor. In vivo studies to test the combination of KPT-330 with Bcl-2 inhibitors are ongoing. Conclusions: We introduce a new therapeutic approach for RCC treatment based on the inhibition of the nuclear export of key tumor suppressors. Inhibition of CRM1 causes forced nuclear retention, and thereby activation, of several key p53-pathway proteins, leading to apoptosis in RCC cell lines in vitro and tumor growth inhibition in vivo.

scholarly journals Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia

Blood ◽  
2012 ◽  
Vol 120 (9) ◽  
pp. 1765-1773 ◽  
Author(s):  
Parvathi Ranganathan ◽  
Xueyan Yu ◽  
Caroline Na ◽  
Ramasamy Santhanam ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Nuclear Export ◽  
Myeloid Leukemia ◽  
Fold Increase ◽  
In Vivo Studies ◽  
Ic50 Values ◽  
Acute Myeloid

AbstractChromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (eg, p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD–positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

scholarly journals The Mechanisms By Which Mutant-NPM1 Uncouples Differentiation from Proliferation Are Reversed By Several Drugs, Enabling Rational Multi-Component Non-Cytotoxic Differentiation Therapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 878-878
Author(s):  
Xiaorong Gu ◽  
Quteba Ebrahem ◽  
Reda Z. Mahfouz ◽  
Metis Hasipek ◽  
Francis Enane ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Nuclear Export ◽  
Molecular Mechanisms ◽  
Terminal Differentiation ◽  
Hek293 Cells ◽  
Monocytic Differentiation ◽  
Cell Nuclei

Abstract NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML). Unfortunately, there are no 'precision' or rational treatments for this subtype of AML.To elucidate molecular mechanisms of pathogenesis, we performed the first comprehensive, unbiased analysis of the endogenous NPM1 protein-interactome using mass-spectrometry (LC-MS/MS). This approach identified abundant amounts of the master transcription factor driver of monocyte lineage-differentiation PU.1 (SPI1). The NPM1/PU.1 interaction causes PU.1 functional deficiency when NPM1 is mutated, because mutant-NPM1 dislocates PU.1 into the cytoplasm with it.This was confirmed using six different methods: (i) Immunoprecipitation (IP)-LC-MS/MS from nuclear and cytoplasmic fractions of wildtype (wt) and NPM1 -mutated AML cell lines (n=2); (ii) IP-Western blot (WB) from nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=2); (iii) WB of nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=5); (iv) immunofluorescence microscopy (IF) of NPM1 -mutated/wt AML cell lines (n=5); (v) IF of NPM1 -mutated/wt primary AML cells (n=6); and (vi) cotransfection of HEK293 cells to express PU.1 + mutant vs wt-NPM1 followed by IF. Re-introduction of Pu.1 into the nucleus of Pu.1-null myeloid precursors which are differentiation-arrested and exponentially proliferating repressed key myeloid precursor genes (e.g., Hoxa9) and triggered terminal monocytic differentiation. Even though primary AML cells (n=900) express the PU.1/RUNX1/CEBPA monocyte differentiation-driving master transcription factor circuit at levels comparable to or exceeding that in normal monocytes, the expression of ~300 monocyte terminal-differentiation genes was suppressed. Importantly, the genes affected are strongly positively correlated (avg. rho 0.7) with PU.1 expression in normal hematopoiesis, consistent with functional disruption of PU.1 in AML. To translate these observations into a treatment option for NPM1 -mutated AMLs, we were guided by additional observations. First, the NPM1/PU.1 protein complex is exported by the nuclear export protein XPO1. XPO1-mediated nuclear export is inhibited by the small molecule selinexor. We found that sub-cytotoxic /low nanomolar concentrations of selinexor locked mutant-NPM1/PU.1 in the nucleus, releasing terminal monocytic differentiation of NPM1 -mutated AML cells both in vitro and in vivo . Briefly, NSG mice were xenotransplanted with NPM1 / FLT3 -mutated primary AML cells and observed until AML engraftment (≥20%) was confirmed. Selinexor was then administered by oral gavage at 2 mg/kg 4X/week (Fig1A), which is 10-fold lower than the usual in vivo cytotoxic dose (15-20 mg/kg) - low doses are well-tolerated and sufficient to promote non-cytotoxic differentiation of AML cells. After 50 days of treatment, bone marrow (Fig1B) and spleen (Fig1C) AML burden was significantly lower in selinexor vs vehicle treated mice. In addition, selinexor treated mice preserved murine hematopoiesis (Fig1D) and IF confirmed the partial nuclear restoration of PU.1 (Fig1E). Terminal monocytic differentiation of AML cells was evident by Giemsa-stained morphology (Fig1F) and flow cytometry (Fig1G). RUNX1 and CEBPA remain in NPM1 -mutated AML cell nuclei at high levels - PU.1 usually cooperates with these master transcription factor partners to exchange corepressors for coactivators and activate differentiation genes. Accordingly, IP-LC-MS/MS of endogenous nuclear CEBPA demonstrated enrichment for corepressors. Depletion of one of these corepressors, DNMT1, using non-cytotoxic concentrations of decitabine or 5-azacytidine (clinical DNMT1-depletors), also induced terminal-differentiation. Moreover, the granulocytic direction of differentiation naturally downregulated NPM1, an event inherent to CEBPA-driven granulocytic (but not monocytic) differentiation. This approach readily induced terminal-differentiation in NPM1 -mutated AML cells selected over 52 weeks of culture for resistance to selinexor, shown by exponential growth in selinexor 20 nM. The mechanisms by which mutant-NPM1 creates leukemic self-replication (proliferation uncoupled from differentiation) are thus reversed by non-cytotoxic molecular targeted clinical drugs. We are evaluating the combination of selinexor with non-cytotoxic DNMT1-depletion in vivo and clinical trials are planned. Disclosures Landesman: Karyopharm Therapeutics: Employment.

CRM1 As a New Therapeutic Target For Non-Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1300-1300
Author(s):  
Yuankai Shi ◽  
Jianfei Wang ◽  
Xiaohong Han ◽  
Ningning Zhang ◽  
Shuai Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Nuclear Export ◽  
Conflicts Of Interest ◽  
Human Lymphocytes ◽  
Xenograft Model ◽  
Lymphoma Cells

Abstract Background The nuclear export protein chromosomal region maintenance 1 (CRM1) may play a role in human neoplasia and serve as a novel target for cancer treatment. Investigators recently developed orally bioavailable selective inhibitors of nuclear export (SINEs) that irreversibly bind to CRM1 and block its function. Our objective was to evaluate the therapeutic efficacy of the novel SINEs KPT-185 and KPT-276 against NHL in vitro and in vivo and elucidate the mechanism of CRM1 inhibitor-mediated antitumor activity. Methods Cell viability, apoptosis and cell cycle were evaluated in 8 B and T cell lymphoma cell lines (Jeko-1, Mino, Granta519, Sp53, RL, Hut102, Hut78 and Jurkat) which were treated with KPT-185; Primary tumor cells from 5 patients and normal human lymphocytes from 2 healthy volunteers were treated directly with KPT-185. Tumor suppressor proteins were detected by western blot to explore the possible mechanisms of KPT-185 inducing lymphoma cells growth inhibition and apoptosis. BALB/c nude mice bearing Jeko-1 tumors were treated orally with KPT-276 (similar structure to KPT-185, but improved animal pharmacokinetics) to examine the efficacy and side-effects of KPT-276. Results KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (half-maximal inhibitory concentrations, 60-120 nM) and induced cell-cycle arrest and apoptosis in NHL cell lines and normal lymphocytes. The antitumor activity mainly consisted of regulating cell growth and apoptosis mechanisms by inducing caspase cleavage and downregulating the expression of antiapoptotic proteins such as CRM1, nuclear factor-kB, and survivin. Furthermore, oral administration of KPT-276 significantly suppressed tumor growth and prolonged survival in mice with NHL xenografts without any major toxic effects (P < 0.001). Analysis of tumor remnants in the mice demonstrated that KPT-276 trapped the antiapoptoic protein survivin within the nuclei of NHL cells. Conclusions We observed the biologic and pharmacologic activity of CRM1-inhibiting SINEs in NHL cells, primary NHL tumor samples, and a murine NHL xenograft model. SINE CRM1 inhibitors inhibited growth of lymphoma cells both in vitro and in vivo. The antitumor activity of the SINEs resulted primarily from induction of caspase activity and downregulation of expression of antiapoptoic proteins such as survivin and NF-kB. The preclinical in vitro and in vivo results reported herein support further study of CRM1-inhibiting SINEs as novel therapeutics for NHL. Disclosures: No relevant conflicts of interest to declare.

Evaluation of selective inhibitors of nuclear export (SINE) CRM1 inhibitors for the treatment of renal cell carcinoma (RCC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 4634-4634 ◽  
Author(s):  
Hiromi Inoue ◽  
Michael Kauffman ◽  
Sharon Shacham ◽  
Yosef Landesman ◽  
Robert H Weiss
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Nuclear Export ◽  
Kinase Inhibitors ◽  
Chromosome Region ◽  
Human Kidney ◽  
Tumor Growth Inhibition ◽  
Novel Therapeutic

4634 Background: For the ~30% of patients who present with RCC at the metastatic stage, multi-kinase inhibitors have been used with moderate success: progression-free survival remains at only one to two years, and thus it is imperative to discover novel therapeutic approaches for metastatic disease. We asked whether (1) SINE inhibitors of chromosome region maintenance protein 1 (CRM1) attenuate key cell cycle regulatory and apoptotic molecules and whether these compounds exert salutary effects in a human RCC xenograft mouse model. Methods: Four RCC cell lines (ACHN, Caki-1, 786-O, and A498) with distinct genotypes, and primary normal human kidney (NHK) cell lines, were used in this study. The cells were treated with the chemically related SINE CRM1 inhibitors KPT-185 or 251 and MTT assays were performed. In addition, cell cycle analyses, immunofluorescence for p53 and p21, and immunoblotting for CRM1, p53, p21, p27, and p-MDM2 were performed for all cell lines. RCC mice with Caki-1 xenografts were treated with vehicle, the orally-available CRM1 inhibitor KPT-251, or sorafenib for 26 days. Tumor volume was measured over several days. Results: Both KPT185 and 251 specifically reduced CRM1 protein levels in RCC cells. KPT-185 caused dose-dependent cytotoxicity in RCC cells, which was greater than sorafenib in RCC cell lines but less in NHK cells, suggesting a possible clinical advantage of KPT-185 over sorafenib. By FACS analysis, we showed that KPT-185 arrests the cell cycle in both G2/M and G1, and increased the sub-G0 cell population. KPT-185 and 251 both increased p53 and p21 in RCC cells, and KPT-185 confined these proteins to the nucleus. In vivo, KPT-251 inhibited Caki-1 xenografts in mice compared to both vehicle and sorafenib without obvious systemic adverse effects. Conclusions: We introduce a completely novel therapeutic approach to the treatment of RCC based on inhibition of the nuclear export of key cell cycle regulatory proteins. Inhibition of CRM1 leads to forced nuclear retention, and thereby activation, of several key p53-pathway proteins, leading to cell cycle arrest and apoptosis in RCC cell lines in vitro and tumor growth inhibition in vivo.

scholarly journals MDM2 Inhibition in Combination with Endocrine Therapy and CDK4/6 Inhibition for the Treatment of ER-Positive Breast Cancer

2020 ◽  
Author(s):  
Neil Portman ◽  
Heloisa H. Milioli ◽  
Sarah Alexandrou ◽  
Rhiannon Coulson ◽  
Aliza Yong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Endocrine Therapy ◽  
Cell Lines ◽  
Patient Derived Xenograft ◽  
Er Positive ◽  
Mcf 7 ◽  
Positive Breast Cancer

AbstractBackgroundResistance to endocrine therapy is a major clinical challenge in the management of estrogen receptor (ER)-positive breast cancer. In this setting p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment resistant ER-positive breast cancer.MethodsWe used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in p53 wildtype and p53 mutant ER positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant ER positive breast cancer.ResultsWe demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant resistant patient derived xenograft model.ConclusionsWe conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programs.

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