Melphalan and Exportin 1 Inhibitors Exert Synergistic Antitumor Effects in Preclinical Models of Human Multiple Myeloma.

High-dose chemotherapy with melphalan followed by autologous transplantation is a first-line treatment for multiple myeloma. Here, we present preclinical evidence that this treatment may be significantly improved by the addition of exportin 1 inhibitors (XPO1i). The XPO1i selinexor, eltanexor, and KOS-2464 sensitized human multiple myeloma cells to melphalan. Human 8226 and U266 multiple myeloma cell lines and melphalan-resistant cell lines (8226-LR5 and U266-LR6) were highly sensitized to melphalan by XPO1i. Multiple myeloma cells from newly diagnosed and relapsed/refractory multiple myeloma patients were also sensitized by XPO1i to melphalan. In NOD/SCIDγ mice challenged with either parental 8226 or U266 multiple myeloma and melphalan-resistant multiple myeloma tumors, XPO1i/melphalan combination treatments demonstrated stronger synergistic antitumor effects than single-agent melphalan with minimal toxicity. Synergistic cell death resulted from increased XPO1i/melphalan-induced DNA damage in a dose-dependent manner and decreased DNA repair. In addition, repair of melphalan-induced DNA damage was inhibited by selinexor, which decreased melphalan-induced monoubiquitination of FANCD2 in multiple myeloma cells. Knockdown of FANCD2 was found to replicate the effect of selinexor when used with melphalan, increasing DNA damage (γH2AX) by inhibiting DNA repair. Thus, combination therapies that include selinexor or eltanexor with melphalan may have the potential to improve treatment outcomes of multiple myeloma in melphalan-resistant and newly diagnosed patients. The combination of selinexor and melphalan is currently being investigated in the context of high-dose chemotherapy and autologous transplant (NCT02780609). SIGNIFICANCE: Inhibition of exportin 1 with selinexor synergistically sensitizes human multiple myeloma to melphalan by inhibiting Fanconi anemia pathway-mediated DNA repair.

Phase I Clinical Trial of Selinexor in Combination with Daunorubicin and Cytarabine in Previously Untreated Poor-Risk Acute Myeloid Leukemia.

PURPOSE: Induction chemotherapy results in complete remission (CR) rates of 20% to 50% among patients with poor-risk AML. Selinexor is an oral selective inhibitor of nuclear export with promising single-agent activity. By inhibiting the primary export protein, XPO1, selinexor localizes and activates tumor suppressor proteins in the nucleus and inhibits DNA damage repair, rationalizing combination with DNA-damaging agents. PATIENTS AND METHODS: This was a single-arm phase I clinical trial of selinexor combined with cytarabine and daunorubicin (7+3). Dose escalation was selinexor alone (3+3) with an expansion at the MTD. Cohorts 1 and 2 received 60 and 80 mg orally, respectively, twice weekly during induction. Consolidation cycles (≤ 2) with selinexor at induction dose plus 5+2 were allowed for patients who achieved CR. MTD and recommended phase II dose of selinexor were the primary endpoints. RESULTS: Twenty-one patients with poor-risk AML were enrolled. All 21 patients were included in the safety evaluations and survival analyses (4 in each of 2 cohorts; 13 in the expansion); 8 (53%) of the 19 patients evaluable for response achieved CR/CRi. MTD was not reached. Selinexor 80 mg (orally, twice weekly) was used in the expansion phase. The most common grade 3/4 nonhematologic treatment-emergent adverse events were febrile neutropenia (67%), diarrhea (29%), hyponatremia (29%), and sepsis (14%). At median follow-up (28.9 months), 38% of patients were alive. Median overall survival was 10.3 months. CONCLUSIONS: Selinexor plus 7+3 is a safe regimen for patients with newly diagnosed poor-risk AML and warrants further investigation in a larger clinical trial.

XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma.

Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.

Treatment of acquired drug resistance in multiple myeloma by combination therapy with XPO1 and topoisomerase II inhibitors.

BACKGROUND: Acquired drug resistance is the greatest obstacle to the successful treatment of multiple myeloma (MM). Despite recent advanced treatment options such as liposomal formulations, proteasome inhibitors, immunomodulatory drugs, myeloma-targeted antibodies, and histone deacetylase inhibitors, MM is still considered an incurable disease. METHODS: We investigated whether the clinical exportin 1 (XPO1) inhibitor selinexor (KPT-330), when combined with pegylated liposomal doxorubicin (PLD) or doxorubicin hydrochloride, could overcome acquired drug resistance in multidrug-resistant human MM xenograft tumors, four different multidrug-resistant MM cell lines, or ex vivo MM biopsies from relapsed/refractory patients. Mechanistic studies were performed to assess co-localization of topoisomerase II alpha (TOP2A), DNA damage, and siRNA knockdown of drug targets. RESULTS: Selinexor was found to restore sensitivity of multidrug-resistant 8226B25, 8226Dox6, 8226Dox40, and U266PSR human MM cells to doxorubicin to levels found in parental myeloma cell lines. NOD/SCID-γ mice challenged with drug-resistant or parental U266 human MM and treated with selinexor/PLD had significantly decreased tumor growth and increased survival with minimal toxicity. Selinexor/doxorubicin treatment selectively induced apoptosis in CD138/light-chain-positive MM cells without affecting non-myeloma cells in ex vivo-treated bone marrow aspirates from newly diagnosed or relapsed/refractory MM patients. Selinexor inhibited XPO1-TOP2A protein complexes (proximity ligation assay), preventing nuclear export of TOP2A in both parental and multidrug-resistant MM cell lines. Selinexor/doxorubicin treatment significantly increased DNA damage (comet assay/γ-H2AX) in both parental and drug-resistant MM cells. TOP2A knockdown reversed both the anti-tumor effect and significantly reduced DNA damage induced by selinexor/doxorubicin treatment. CONCLUSIONS: The combination of an XPO1 inhibitor and liposomal doxorubicin was highly effective against acquired drug resistance in in vitro MM models, in in vivo xenograft studies, and in ex vivo samples obtained from patients with relapsed/refractory myeloma. This drug combination synergistically induced TOP2A-mediated DNA damage and subsequent apoptosis. In addition, based on our preclinical data, we have initiated a phase I/II study with the XPO1 inhibitor selinexor and PLD (ClinicalTrials.gov NCT02186834). Initial results from both preclinical and clinical trials have shown significant promise for this drug combination for the treatment of MM.

Phase I trial of bortezomib (PS-341; NSC 681239) and "nonhybrid" (bolus) infusion schedule of alvocidib (flavopiridol; NSC 649890) in patients with recurrent or refractory indolent B-cell neoplasms.

PURPOSE: This phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, Waldenstrom macroglobulinemia, and mantle cell lymphoma). EXPERIMENTAL DESIGN: Patients received bortezomib (intravenous push), followed by alvocidib (1-hour infusion), on days 1, 4, 8, and 11 of a 21-day treatment cycle. Patients experiencing responses or stable disease continued on treatment at the investigator's discretion. A standard 3+3 dose-escalation design was used to identify the MTD based on DLTs, and pharmacokinetic and pharmacodynamic studies were conducted. RESULTS: A total of 44 patients were enrolled, with 39 patients assessed for response. The MTD was established as 1.3 mg/m(2) for bortezomib and 40 mg/m(2) for alvocidib. The most common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. The most common nonhematologic toxicities included diarrhea, fatigue, and sensory neuropathy. Three complete remissions (8%) and 10 partial remissions (26%) were observed for a total response rate of 33%. Pharmacokinetic findings with the current dosing regimen were consistent with the comparable literature and the hybrid dosing regimen. Pharmacodynamic study results did not correlate with clinical responses. CONCLUSIONS: The combination of bortezomib and alvocidib is tolerable, and an MTD has been established for this schedule. The regimen appears to be efficacious in patients with relapsed/refractory multiple myeloma or indolent non-Hodgkin lymphoma. As the nonhybrid regimen is less cumbersome than the previous hybrid dosing schedule regimen, the current schedule is recommended for successor studies.

Inhibition of CRM1-dependent nuclear export sensitizes malignant cells to cytotoxic and targeted agents.

Nuclear-cytoplasmic trafficking of proteins is a significant factor in the development of cancer and drug resistance. Subcellular localization of exported proteins linked to cancer development include those involved in cell growth and proliferation, apoptosis, cell cycle regulation, transformation, angiogenesis, cell adhesion, invasion, and metastasis. Here, we examined the basic mechanisms involved in the export of proteins from the nucleus to the cytoplasm. All proteins over 40kDa use the nuclear pore complex to gain entry or exit from the nucleus, with the primary nuclear export molecule involved in these processes being chromosome region maintenance 1 (CRM1, exportin 1 or XPO1). Proteins exported from the nucleus must possess a hydrophobic nuclear export signal (NES) peptide that binds to a hydrophobic groove containing an active-site Cys528 in the CRM1 protein. CRM1 inhibitors function largely by covalent modification of the active site Cys528 and prevent binding to the cargo protein NES. In the absence of a CRM1 inhibitor, CRM1 binds cooperatively to the NES of the cargo protein and RanGTP, forming a trimer that is actively transported out of the nucleus by facilitated diffusion. Nuclear export can be blocked by CRM1 inhibitors, NES peptide inhibitors or by preventing post-translational modification of cargo proteins. Clinical trials using the classic CRM1 inhibitor leptomycin B proved too toxic for patients; however, a new generation of less toxic small molecule inhibitors is being used in clinical trials in patients with both hematological malignancies and solid tumors. Additional trials are being initiated using small-molecule CRM1 inhibitors in combination with chemotherapeutics such as pegylated liposomal doxorubicin. In this review, we present evidence that combining the new CRM1 inhibitors with other classes of therapeutics may prove effective in the treatment of cancer. Potential combinatorial therapies discussed include the use of CRM1 inhibitors and the addition of alkylating agents (melphalan), anthracyclines (doxorubicin and daunomycin), BRAF inhibitors, platinum drugs (cisplatin and oxaliplatin), proteosome inhibitors (bortezomib and carfilzomib), or tyrosine-kinase inhibitors (imatinib). Also, the sequence of treatment may be important for combination therapy. We found that the most effective treatment regimen involved first priming the cancer cells with the CRM1 inhibitor followed by doxorubicin, bortezomib, carfilzomib, or melphalan. This order sensitized both de novo and acquired drug-resistant cancer cell lines.

Rapid screening of novel agents for combination therapy in sarcomas.

For patients with sarcoma, metastatic disease remains very difficult to cure, and outcomes remain less than optimal. Treatment options have not largely changed, although some promising gains have been made with single agents in specific subtypes with the use of targeted agents. Here, we developed a system to investigate synergy of combinations of targeted and cytotoxic agents in a panel of sarcoma cell lines. Agents were investigated alone and in combination with varying dose ratios. Dose-response curves were analyzed for synergy using methods derived from Chou and Talalay (1984). A promising combination, dasatinib and triciribine, was explored in a murine model using the A673 cell line, and tumors were evaluated by MRI and histology for therapy effect. We found that histone deacetylase inhibitors were synergistic with etoposide, dasatinib, and Akt inhibitors across cell lines. Sorafenib and topotecan demonstrated a mixed response. Our systematic drug screening method allowed us to screen a large number of combinations of sarcoma agents. This method can be easily modified to accommodate other cell line models, and confirmatory assays, such as animal experiments, can provide excellent preclinical data to inform clinical trials for these rare malignancies.

CRM1 Inhibition Sensitizes Drug Resistant Human Myeloma Cells to Topoisomerase II and Proteasome Inhibitors both In Vitro and Ex Vivo.

Multiple myeloma (MM) remains an incurable disease despite improved treatments, including lenalidomide/pomalidomide and bortezomib/carfilzomib based therapies and high-dose chemotherapy with autologous stem cell rescue. New drug targets are needed to further improve treatment outcomes. Nuclear export of macromolecules is misregulated in many cancers, including in hematological malignancies such as MM. CRM1 (chromosome maintenance protein-1) is a ubiquitous protein that exports large proteins (>40 kDa) from the nucleus to the cytoplasm. We found that small-molecule Selective Inhibitors of Nuclear Export (SINE) prevent CRM1-mediated export of p53 and topoisomerase IIα (topo IIα). SINE's CRM1-inhibiting activity was verified by nuclear-cytoplasmic fractionation and immunocytochemical staining of the CRM1 cargoes p53 and topo IIα in MM cells. We found that SINE molecules reduced cell viability and induced apoptosis when used as both single agents in the sub-micromolar range and when combined with doxorubicin, bortezomib, or carfilzomib but not lenalidomide, melphalan, or dexamethasone. In addition, CRM1 inhibition sensitized MM cell lines and patient myeloma cells to doxorubicin, bortezomib, and carfilzomib but did not affect peripheral blood mononuclear or non-myeloma bone marrow mononuclear cells as shown by cell viability and apoptosis assay. Drug resistance induced by co-culture of myeloma cells with bone marrow stroma cells was circumvented by the addition of SINE molecules. These results support the continued development of SINE for patients with MM.

Nuclear export of proteins and drug resistance in cancer.

The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21(CIP), p27(KIP1), N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer.

Phase I Study of Topotecan, Ifosfamide, and Etoposide (TIME) with autologous stem cell transplant in refractory cancer: pharmacokinetic and pharmacodynamic correlates.

PURPOSE: To determine the maximum tolerated dose (MTD) of topotecan in combination with ifosfamide, mesna, and etoposide (TIME), followed by autologous hematopoietic cell transplant (HCT), in patients with chemotherapy-refractory malignancies. EXPERIMENTAL DESIGN: Patients were treated with (in mg/m(2)/d) ifosfamide 3,333, mesna 3,333, and topotecan 3.3 to 28.3 during days -8 through -6 and etoposide 500 (days -5 through -3) followed by HCT on day 0. Once MTD was defined, we expanded this dosing cohort to include patients with high-risk lymphoma due to activity seen during dose escalation. Topotecan pharmacokinetic analyses were carried out, and topoisomerase I levels and activity were measured. RESULTS: The topotecan MTD in this regimen was 64 mg/m(2) (21.3 mg/m(2)/d). Mucositis was dose limiting and correlated with topotecan dose level and area under the curve (AUC). Dose level was also correlated with length of hospitalization, number of days of parenteral nutrition, and neutrophil and platelet engraftment. Topotecan AUC was significantly correlated with time to platelet recovery. The baseline peripheral blood mononuclear cell topoisomerase I level was found to be a significant positive predictor for overall and progression-free survival. Topotecan AUC was positively correlated with dose level, with a trend toward decreasing clearance with increasing dose. CONCLUSION: Topotecan can be a useful drug in the high-dose setting given its activity in some malignancies when given in standard dose. Pharmacokinetic monitoring may be a valuable tool for optimizing the use of topotecan and to avoid toxicity seen with high-systemic exposures. Baseline topoisomerase I levels may have an important role in predicting topotecan efficacy.

Phase I trial of bortezomib (PS-341; NSC 681239) and alvocidib (flavopiridol; NSC 649890) in patients with recurrent or refractory B-cell neoplasms.

PURPOSE: A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, and mantle cell lymphoma). EXPERIMENTAL DESIGN: Patients received bortezomib by intravenous push on days 1, 4, 8, and 11. Patients also received alvocidib on days 1 and 8 by 30-minute bolus infusion followed by a 4-hour continuous infusion. Treatment was on a 21-day cycle, with indefinite continuation for patients experiencing responses or stable disease. Dose escalation employed a standard 3 + 3 design until the MTD was identified on the basis of DLTs. Pharmacokinetic studies and pharmacodynamic studies were conducted. RESULTS: Sixteen patients were treated. The MTD was established as 1.3 mg/m(2) for bortezomib and 30 mg/m(2) for alvocidib (both the 30-minute bolus and 4-hour infusions). Common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Common nonhematologic toxicities included fatigue and febrile neutropenia. DLTs included fatigue, febrile neutropenia, and elevated aspartate aminotransferase (AST) levels. Two complete responses (CR; 12%) and five partial responses (PR; 31%) were observed at the MTD (overall response rate = 44%). Pharmacokinetic results were typical for alvocidib and pharmacodynamic studies yielded variable results. CONCLUSIONS: The combination of bortezomib and alvocidib is tolerable and an MTD has been established for the tested schedule. The regimen appears active in patients with relapsed and/or refractory multiple myeloma or non-Hodgkin's lymphoma, justifying phase II studies to determine the activity of this regimen more definitively.

Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition.

Topoisomerase IIalpha (topo IIalpha) is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism at cellular densities similar to those found in patient bone marrow. When topo IIalpha is trafficked to the cytoplasm, it is not in contact with the DNA; thus, topo IIalpha inhibitors are unable to induce DNA-cleavable complexes and cell death. Using a CRM1 inhibitor or a CRM1-specific small interfering RNA (siRNA), we were able to block nuclear export of topo IIalpha as shown by immunofluorescence microscopy. Human myeloma cell lines and patient myeloma cells isolated from bone marrow were treated with a CRM1 inhibitor or CRM1-specific siRNA and exposed to doxorubicin or etoposide at high cell densities. CRM1-treated cell lines or myeloma patient cells were 4-fold more sensitive to topo II poisons as determined by an activated caspase assay. Normal cells were not significantly affected by CRM1-topo II inhibitor combination treatment. Cell death was correlated with increased DNA double-strand breaks as shown by the comet assay. Band depletion assays of CRM1 inhibitor-exposed myeloma cells showed increased topo IIalpha covalently bound to DNA. Topo IIalpha knockdown by a topo IIalpha-specific siRNA abrogated the CRM1-topo II therapy synergistic effect. These results suggest that blocking topo IIalpha nuclear export sensitizes myeloma cells to topo II inhibitors. This method of sensitizing myeloma cells suggests a new therapeutic approach to multiple myeloma.

Potentiation of a topoisomerase I inhibitor, karenitecin, by the histone deacetylase inhibitor valproic acid in melanoma: translational and phase I/II clinical trial.

PURPOSE: The novel topoisomerase I inhibitor karenitecin (KTN) shows activity against melanoma. We examined whether histone deacetylase inhibition could potentiate the DNA strand cleavage, cytotoxicity as well as the clinical toxicity, and efficacy of KTN in melanoma. EXPERIMENTAL DESIGN: Apoptosis, COMET, and xenograft experiments were carried out as described previously. A phase I/II trial of valproic acid (VPA) and KTN was conducted in patients with stage IV melanoma, with any number of prior therapies, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. RESULTS: VPA pretreatment potentiated KTN-induced apoptosis in multiple melanoma cell lines and in mouse A375 xenografts. VPA increased KTN-induced DNA strand breaks. In the phase I/II trial, 39 patients were entered, with 37 evaluable for toxicity and 33 evaluable for response. Somnolence was the dose-limiting toxicity. The maximum tolerated dose for VPA was 75 mg/kg/d; at maximum tolerated dose, serum VPA was approximately 200 microg/mL (1.28 mmol/L). At the dose expansion cohort, 47% (7 of 15) of patients had stable disease; median overall survival and time to progression were 32.8 and 10.2 weeks, respectively. Histone hyperacetylation was observed in peripheral blood mononuclear cells at maximum tolerated dose. CONCLUSION: VPA potentiates KTN-induced DNA strand breaks and cytotoxicity. VPA can be combined at 75 mg/kg/d for 5 days with full-dose KTN without overlapping toxicities. In metastatic poor prognosis melanoma, this combination is associated with disease stabilization in 47% of patients. Further testing of this combination appears warranted.