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Publications / Abstracts


American Society of Clinical Oncology (ASCO) Annual Meeting
Chicago, USA
June, 2012

Preclinical evaluation of selective inhibitors of nuclear export (SINE) in basal-like breast cancer (BLBC).

Dilara McCauley 1, Yosef Landesman1, William Senapedis1, Trinayan Kashyap1, Jean-Richard Saint-Martin1, Louis Plamondon1, Vincent Sandanayaka1, Sharon Shechter1, Doriana Froim1, Raphael Nir2, Jennifer Williams2, Lynda Chin, Cyril Benes, Mansoor Raza Mirza6, Michael Kauffman1, Sharon Shacham1;

  1. Karyopharm Therapeutics, Natick, MA;
  2. SBH Sciences, Natick, MA;
  3. Institute for Applied Cancer Science, University of Texas M. D. Anderson Cancer Center, Houston, TX;
  4. Massachusetts General Hospital Cancer Center, Boston, MA;
  5. NSGO-Nordic Society of Gynaecological Oncology, Copenhagen, Denmark

Abstract:

Background:Basal-like breast cancers (BLBC) compose up to 15% of breast cancer (BC) and are usually triple negative characterized by lack of ER, PR, and HER-2 amplification. In addition, most BRCA1-associated BCs are BLBC and TNBC, expressing basal cytokeratins and EGFR. BLBC is characterized by an aggressive phenotype, high histological grade, and poor clinical outcomes: high recurrence and metastasis rates. CRM1 (XPO1) is the exclusive nuclear exporter of multiple Tumor Suppressor Proteins (TSP) including p53, p21, BRCA1&2, pRB, FOXO. CRM1 inhibition forces nuclear accumulation of TSPs, inducing apoptosis in cancer cells. KPT-SINE are novel, small molecule, irreversible inhibitors of CRM1 with potent anti cancer activity.

Methods: The Cancer Genome Atlas (TCGA) and BC cell line databases were used for mRNA analyses. MTT assay was used to determine the cytotoxic effect of KPT-SINE (KPT-185 and KPT-330) on 44 breast cell lines including luminal A, luminal B, HER2 positive, BLBC and TNBC cells. The effect of KPT-330 treatment on tumor growth was tested in vivo in the TNBC model MDA-MB-468 xenograft.

Results: Analyses of nuclear pore complex (NPC)-related mRNA levels (including CRM1) showed clear separation of BCs into high and low NPC expression. BLBC subtype was enriched with high NPC transcripts while luminal BC was enriched in low NPC levels (p<1.53e-20). High NPC levels had higher mutation levels in BRCA2 (cor=0.33, p=1.83e-8) and ABL1. NPC expression was inversely correlated with ER mRNA expression (cor=-0.58, p=1.37e-7). KPT-SINE showed potent cytotoxicity on >75% of the cell lines (IC50 values <1 μM). Only three of 24 TNBC cell lines displayed IC50 values >1.5 μM upon KPT-SINE treatment. Genomic analyses on all BC lines indicated that p53, PI3K/AKT and BRCA1 or 2 status did not affect cytotoxicity. In MDA-MB-468 xenograft, KPT-330 displayed efficacy in a dose-dependent manner inhibiting nearly 100% of tumor growth compared with vehicle treated animals, and was well tolerated.

Conclusions: These data show that NPC/CRM1 mRNAs are overexpressed in BLBC/TNBC and that CRM-1 mediated nuclear export inhibition by SINE represents a potentially novel and well tolerated therapy for BLBC / TNBC.

 



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American Association for Cancer Research (AACR) Meeting
Chicago, USA
April 2, 2012

Selective inhibitors of nuclear export (SINE) induce multiple tumor suppressor proteins (TSP) activity and show single agent antitumor effect and synergy with Bcl-2 antagonist in non-small cell lung cancer

Dilara McCauley 1, Tami Rashal2, Sharon Shacham1, Michael Kauffman1, Yossi Landesman1, Jean St-Martin1, Trinayan Kashyap1, William Senapedis1, Jennifer L. Williams3, Raphael Nir3, Maya Illuz2, Nir Peled2.

  1. Karyopharm Therapeutics, Natick, MA
  2. Sheba Medical Center, Tel Hashomer, Israel
  3. SBH Sciences, Natick, MA

Neoplastic cells must inactivate their tumor suppressor proteins (TSP) in order to perpetuate their growth. Oncogene and growth factor driven nuclear export of TSPs is an increasingly recognized mechanism for TSP inactivation. We have developed orally active, small molecule SINE that irreversibly block the major nuclear export protein CRM1 (exportin 1, XPO1) and selectively induce the death of cancer cells. Forced nuclear accumulation of TSPs is believed to initiate a "genome survey" leading to the death of cancer cells, whereas normal cells undergo transient, reversible cell cycle arrest. We show that the small molecule SINE CRM1 inhibitor KPT-185 potently kills many types of tumor cells in vitro (IC50 20-500nM, IC80 ≤ 1µM at 72 hours) including ~75% of NSCLC lines with diverse genetic signatures (EGFR wt & mut, p53 wt & mut, K-ras wt & mut). In contrast, ~25% of NSCLC lines with similar genotypes show cytotoxicity IC50s ≥ 1.5µM and IC80 is not reached. Treatment of the SINE-resistant A549 NSCLC (adenocarcinoma, EGFR wt, p53 wt, K-ras mut, KPT-185 IC50 = 1.75µM) leads to nuclear localization of p53, p21, FOXO, E2F4, IkB, underphosphorylated pRb and other CRM1 cargoes at concentrations similar to that of sensitive NSCLC cell lines, but apoptosis is not readily induced. To test whether the resistance to CRM1 inhibition-mediated cytotoxicity is due to activation of anti-apoptotic (or inactivation of pro-apoptotic) BCL-2 family proteins, we combined SINE with ABT-737, a small-molecule BCL-2 inhibitor currently in Phase 2 clinical trials, and examined the molecular mechanisms leading to tumor cell death. We show that combination of low doses (500nM, the IC20) of KPT-185 with non-cytotoxic doses of the Bcl-2 inhibitor (≤ 100µM) induces synergistic cytotoxicity. Moreover, the antitumor effects of the combination therapy are manifested within 48 hours, whereas significant cytotoxicity by higher doses of SINE alone requires at least 72 hours. Similar results have been obtained with another SINE resistant cell line, HCC-2935 (EGFR mut, p53 mut, K-ras mut). We noted that in the resistant A549 line, levels of the pro-apoptotic BCL2 member, Bax, were reduced by ~50% with SINE treatment, but that addition of the BCL2 inhibitor reversed this effect and induced significant synergistic cytotoxicity. The sensitive NSCLC cell line H-226 (EGFR wt, p53wt, K-ras wt; IC50 12nM) showed nuclear localization of CRM1 cargoes when treated with KPT-185 (100nM), but only modest additional killing by the addition of the Bcl-2 inhibitor. We conclude that mechanism of resistance to SINE mediated cytotoxicity can be overcome by antagonism of Bcl-2. In vivo studies using A-549 xenografts comparing the effects of KPT-SINE and ABT-737 alone or in combination are ongoing and will be reported at the meeting.



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American Association for Cancer Research (AACR) Meeting
Orlando, USA
April 6, 2011

Discovery of SINE: Selective inhibitors of Nuclear Export increase levels of regulatory proteins p53, p21, FOXO and IkB leading to apoptosis of malignant cells

S. Shacham, M. Kauffman, V. Sandanayaka, G. Draetta, S. Shechter, J. Williams, R. Nir

  • Karyopharm Therapeutics, Newton, MA
  • Dana-Farber Cancer Institute, Boston, MA
  • SBH Sciences, Natick, MA

Transport of macromolecules across nuclear membrane is fundamental to the proper functioning of living cells. Nuclear localization of intact tumor suppressor proteins (TSPs) and other growth regulatory proteins (GRPs) such as p53, FOXO, pRB, p21, p27 and IB are critical for their "policing" function and mislocalization of a nuclear protein into the cytoplasm can render it ineffective as a tumor suppressor protein. During disease progression or in response to the tumor environment, cancer cells appear to acquire intracellular mechanisms to export anti-cancer nuclear proteins, in many cases resulting in proteasome dependent degradation. Most known TSP utilize the nuclear export protein CRM1 (XPO1, chromosomal region maintenance 1), to exit the nucleus, and overexpression of CRM1 has been reported as a poor prognostic factor in a variety of neoplasms. Inhibition of CRM1 can force the nuclear localization of key TSPs, activate their pathways, resulting in induction of cell death of cancer cells (Table 1). SINEs are novel, potent, selective, drug-like, nuclear export inhibitors (EC50 ~40-100nM) that derive their activity through direct covalent modification and inhibition of CRM1. SINE compounds exerts a potent and prolonged inhibition of CRM1-mediated forkhead (FOXO), p53, p21 and IB nuclear export in a variety of normal and transformed cell lines. Cell cycle analysis following incubation with SINE compound, KPT-0127, revealed that it blocks at both the G1/S and G2/M phases, consistent with induction of multiple checkpoint controls. SINE-induced apoptosis is observed in many cancer cell lines (e.g. A375, Jurkat, BL-40, HCT-116), but not in other proliferating cells (e.g. 3T3, mouse embryo fibroblasts). In cytotoxicity assays, SINE compounds showed high potency in both hematologic and solid tumor cell lines (EC50 < 100nM) with limited effect on normal cells (PBMCs, HUVEC, 3T3 EC50 >2000nM).SINE compounds show no significant effect on 37 proteins including several cysteine proteases, minimal CYP inhibition (> 10 M for all major CYPs) and no hERG inhibition. In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (p.o. or s.c.) up to 560 mg/kg, and no deaths were observed. The pharmacokinetics of KPT-0127 is adequate for s.c. and i.v. dosing in vivo. Tumor xenograft studies have been undertaken in colon cancer HCT-116 and myeloma MM.1S tumor bearing mice. SINE induce a potent, dose dependent anti cancer activity in both small (~140mm3) and large (~1400mm3) tumors. All together, these data demonstrate that SINE represents a novel mechanism of action with robust anti tumor activity both in-vitro and in-vivo and promising applications as single agent and for combination therapies.

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2011 Gastrointestinal Cancers
San Fransisco, USA
January 20-22, 2011

Preclinical development of small-molecule CRM1 inhibitors as novel therapy for the treatment of colorectal cancer (CRC).

S. Shacham, M. Kauffman, V. Sandanayaka, G. Draetta, S. Shechter, J. Williams, R. Nir

  • Karyopharm Therapeutics, Newton, MA
  • Dana-Farber Cancer Institute, Boston, MA
  • SBH Sciences, Natick, MA

Background: CRM1 (XPO1) is a key nuclear export protein which controls the location of multiple tumor suppressor (TSP) and growth regulatory (GRP) proteins including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Forced nuclear expression of TSP and GRP by CRM1 inhibition can lead to apoptosis in cancer cells while sparing normal cells.

Methods: Novel small-molecule CRM1 inhibitors were synthesized and nuclear distribution studies were performed in cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in 16 CRC cell lines: LS-123, SW-626, Colo-201, Colo-205, Colo-320DM, Colo-320HSR, Lovo, DLD-1, HCT-15, WiDi, LS-174T, LS-180, SW-620, C2BBe1, HCT-8, HCT-116, and in human peripheral leukocytes (PBMC). Cellular distribution and apoptosis assays were performed on HCT-116. Antitumor activity is assessed in human HCT-116 xenografts in scid-mice.

Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ~300 nM. KPT-0127 is cytotoxic to various CRC cell lines with EC50s of 0.07-1.1 µM; in 9 CRC lines EC50s were < 0.3 mM. In contrast, normal cell lines and PBMCs had EC50 > 5-20 µM. In HCT- 116 cells, KPT-0127 induces cell cycle arrest at both G1/S and G2/M checkpoints and dose dependently increases nuclear p53, followed by an increase in caspase 3. KTP-0127 10µM shows no significant effect on 37 proteins including several cysteine proteases. In mice, KPT-0127 given by SC injection of 30-100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours and is well tolerated. KPT-0127 given SC to mice bearing HCT-116 colon xenografts results in dose-dependent antitumor activity.

Conclusions: The novel small- molecule CRM1 inhibitor KTP-0127 kills CRC lines with multiple TSP, GRP, and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, while sparing normal cells. This likely reflects the ability of CRM1 inhibition to affect multiple critical and non-redundant regulatory pathways. These results support the development of CRM1 inhibitors for the treatment of CRC. IND-enabling CMC and toxicology work are in preparation.

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52nd ASH Annual Meeting and Exposition
Orlando, USA
December 4-7, 2010

Title: Preclinical Development of Small-Molecule CRM1 Inhibitors as Novel Therapy for the Treatment of Myeloma and Other Hematological Malignancies

Sharon Shacham1, Joel Turner2, Raphael Nir3, Giulio Draetta 4, Sharon Shechter1, Vincent Sandanayaka1, Michael Kauffman1 and Daniel Sullivan 2

  1. Karyopharm Therapeutics Inc., Newton, MA
  2. H. Lee Moffitt Cancer Center, Tampa, FL
  3. SBH Sciences Inc., Natick, MA
  4. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA

Background: CRM1 (XPO1) is a key nuclear export protein which controls multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, PI3K/AKT, Wnt/ß-catenin and NF-fB. Further, mislocalization of proteins can abrogate TSP functions and render chemotherapies ineffective. For example, multiple myeloma (MM) cells that are grown at high densities (to mimic the in vivo situation) become resistant to topoisomerase 2 (topo2) inhibitors (e.g., doxorubicin) simply because topo2a is exported from the nucleus. Forcing the nuclear expression of chemotherapy targets, TSP and growth regulatory proteins by CRM1 inhibition can restore drug sensitivity and restore checkpoint control / genome surveying functions. These events lead to apoptosis or autophagy in cancer cells while sparing normal cells. Methods: CRM1 inhibitors were synthesized and nuclear distribution studies were performed in U2OS cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in multiple myeloma (MM), leukemia and lymphoma cell lines and in human peripheral blood mononuclear cells (PBMCs). Toxicology studies were performed in several mouse strains. Antitumor activity is assessed in a xenograft model of human MM.1 cells growing in scid-mice.

Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ~300 nM. KPT-0127 is selectively cytotoxic to various hematological cell lines with EC50s in the 0.02-1.0 µM range, and shows limited cytotoxicity in similar studies in normal cell lines (NIH-3T3, MRC5, HUVECs) and PBMCs (EC50 >5-20 µM). KPT-0127 increases the nuclear localization of IB in HUT-78 leukemia cells and human PBMCs, and inhibits TNF-a secretion in LPS stimulated U937 macrophage derived cells, likely through inhibition of NFB signaling. In MM cells grown at high densities which actively export topo2 to the cytoplasm via CRM1, blocking CRM1-dependent transport strongly enhanced topo2 nuclear localization and augmented the apoptotic effects of doxorubicin in < 24 hours showing clear synergy between KPT-0127 and the anthracycline. Combination of sublethal concentrations of bortezomib plus KPT-0127 in MM1.S and MM.1R myeloma cells (as well as in Jurkat and HS-Sultan) induced synergistic cytotoxicities. In mice, KPT-0127 given by subcutaneous (SC) injection of 30-100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours. In 5 day repeated dose toxicology studies, SC administration of 100 mg/kg KPT-0127 (QD x 5) was well tolerated in mice with no cutaneous or obvious systemic clinical findings. Modest neutrophilia and mild lymphopenia were seen and no neurologic signs resulted from treatment with KPT-0127. Administration of KPT-0127 SC to mice bearing HCT-116 colon cancer results in dose-dependent antitumor activity. Xenograft studies with MM.1 myeloma cells are underway and will be presented at the meeting.

Conclusions: The sensitivity of tumors with multiple TSP and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, to killing with KPT-0127 likely reflects the ability to affect multiple critical and non-redundant regulatory pathways. CRM1 inhibition also forces topo 2 to remain in the nucleus, and increases levels of nuclear IkB antagonizing NF-kB function, thereby reducing the likelihood of resistance development. These results support the development of small molecule, drug-like CRM1 inhibitors for the treatment of MM and other hematological cancers. IND-enabling CMC and toxicology work are expected to begin in early 2011.

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XXXIII World Congress of the International
Society of Hematology (ISH 2010)
Jerusalem, Israel
October 10-13, 2010

KPT0127, A novel, potent and selective inhibitor of CRM-1Mediated Nuclear export shows selective cytotoxicity for the treatment of hematological cancers

S. Shacham1, R. Nir2, D. Daelemans3, S. Shechter1, I. Vancurova4, A. Juvekar4, J. Turner5, W. Lau6, G. Higgins6, G. Draetta7, M. Kauffman1

  1. Karyopharm Therapeutics, Newton, MA, USA
  2. SBH Sciences, Natick, MA, USA
  3. Rega Institute for Medical Research, Leuven, Belgium
  4. St.John's University, New York, NY, USA
  5. Moffitt Cancer Center, Tampa, FL, USA
  6. Cancog Technologies, Toronto, ON, Canada
  7. DanaFarber Cancer Institute, Boston, MA, USA

CRM1 (Xpo1) is the major export factor for proteins from the nucleus to the cytoplasm, including tumor suppressors (TSPs) and other modulators of proliferative responses such as p53, FOXO, pRB, Topoisomerase IIA and IkB. Leptomycin B (LMB), a non-drug like natural product is a potent inhibitor of CRM1. LMB demonstrated potent anti-cancer activity in vitro with limited efficacy in vivo due to severe gastrointestinal toxicity. Our lead compound KPT-0127, is a novel, small molecule, water soluble, drug-like, selective, irreversible CRM1 antagonist. KPT-0127 exerts potent (IC50 300-400nM) inhibition of CRM1-mediated HIV-1 Rev, FOXO and p53 nuclear export. In cytotoxicity assays, KPT-0127 showed high potency in most hematologic cancer cell lines (EC50 20-500nM) with limited effect on normal cells (EC50 >5-10µM). Treatment of human myeloma cell lines with KPT-0127 and doxorubicin at high cell densities resulted in >5-fold increased sensitivity to topoisomerase II inhibitors as determined by an activated caspase assay. In normal peripheral blood mononuclear cells (PBMCs) and in Hut78 leukemia cells, KPT-0127 potently increased the nuclear levels of IkB. However, KPT-0127 induced cell death of Hut78 cells with no effect on normal PBMCs. In combination studies, KPT-0127 showed additive or synergistic cytotoxicity activity with either 5-FU, carboplatin, or doxorubicin. KPT-0127 was well tolerated in acute and 5 day, once daily repeated dose mouse toxicology studies. In vivo efficacy studies are currently being performed and will be presented. Together, these data demonstrate that KPT-0127 represents a novel, tumor selective and well-tolerated irreversible Crm1 as a novel therapy for hematological cancers.

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American Chemical Society Meeting
Boston, USA
August 22, 2010

Discovery of CRM1- Mediated Nuclear Transport Inhibitors as Novel Modulators of Epigenetic Enzymes.

Vincent Sandanayaka, Raphael Nir, Sharon Shechter, Michael Kauffman and Sharon Shacham

Karyopharm Therapeutics, 262 Arnold Rd, Newton, MA 02459, USA

Transport of macromolecules across nuclear membrane is fundamental to the proper functioning of living cells (Figure I). Molecular transport between the nucleus and the cytoplasm through the nuclear pore complex is mediated by transport receptors called karyopherins. One such well-recognized receptor is CRM1 (XPO1, chromosomal region maintenance 1), which is essential to the nuclear export of various "cargo" proteins (e.g., p53, c-Abl, IkB, FOXO) and epigenetic histone deacetylases (HDACs) including HDACs 1, 3, 4, 5, 6, 7 and SIRT proteins. Moreover, histone acetyl-transferase (HAT) and HDACs modulate p53, NFkB and AKT pathways. Therefore, inhibition of CRM1 may alter the epigenetic activity of HDAC and HAT. Abnormal CRM1 expression is well correlated with the prognosis of various human cancers, including pancreas, glioma, liver, ovarian, cervical and osteosarcoma. Moreover, CRM1 has been validated in animal models as a novel target for cancer. CRM1 inhibitors such as Leptomycin B (LMB) are efficacious in xenografts, but exhibit dose limiting toxicities that have prevented successful testing in human clinical trials (Figure II). Karyopharm Therapeutics is taking the lead in this field to discover small molecule CRM1 inhibitors that are devoid of toxicities seen with other compounds. Employing the power of in-silico screening and structural biology, we discovered a variety of chemotypes with potent CRM1 inhibition. We will describe the design of several chemotypes using our integrated computational and medicinal chemistry strategies, and their biological activities. Our compounds have shown CRM1-mediated HIV-rev (IC50 = 360 nM), FOXO, IkB and p53 nuclear export inhibition similar to LMB. In addition, our small molecule irreversible inhibitors were shown to act at the CRM1 Cys528 position, and exhibited potent cytotoxicity in hematologic and solid tumor cancer cell lines. We will describe the design of several chemotypes using our integrated computational and medicinal chemistry strategies and their biological activities. Our compounds showed CRM1-mediated HIV-rev nuclear export inhibition similar to LMB (IC50 = 360 nM), CRM1-mediated FOXO nuclear export inhibition in U2OS cells (IC50 = 276 nM), and exhibited potent cytotoxicity in hematologic and solid tumor cancer cell lines. Our data indicate that our irreversible compounds specifically modify CYS528 of CRM1, as a CYS528SER mutant CRM1, which retains full export activity, is not inhibited with our compounds. Pharmacokinetic and in-vivo efficacy studies are on-going and results will be presented at the meeting.
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AACR Translational Cancer Medicine 2010
San Francisco, USA
July 11 – 14, 2010.


KPT0127, A Novel, Potent and Selective Inhibitor of Crm1-Mediated Nuclear Export Shows Selective Cytotoxicity for Neoplastic over Normal Cells and is Well Tolerated in Animals

Sharon Shacham1, Raphael Nir2, Dirk Daelemans3, Sharon Shechter1, Ivana Vancurova4, Ashish Juvekar4, Winnie Lau5, Guy Higgins5, Giulio Draetta6 and Michael Kauffman1

  1. Karyopharm Therapeutics, 262 Arnold Rd, Newton, MA
  2. SBH Sciences, Natick, MA
  3. Rega Institute for Medical Research, Leuven, Belgium
  4. St. John's University 8000 Utopia Parkway New York 11439
  5. Cancog Technologies, Toronto, Canada
  6. Dana Farber Cancer Institute, Boston, MA

CRM1 (Xpo1) is the major export factor for proteins from the nucleus to the cytoplasm, including tumor suppressors (TSPs) and other modulators of proliferative responses such as p53, FOXO, c-Abl, pRB and IB. Leptomycin B (LMB), a non-drug like natural product is a potent inhibitor of CRM1-mediated nucleocytoplasmic transport. LMB and related analogs trap TSPs and other proteins in the nucleus, forcing neoplastic cells into apoptosis while normal cells undergo reversible cell cycle arrest. However, LMB has limited efficacy in vivo due to its severe gastrointestinal toxicity. Here, we describe our lead compound KPT-0127 a novel small molecule, water soluble, drug-like, selective, irreversible CRM1 antagonist. Like LMB, KPT-0127 forms a covalent bond with Cys-528 in the CRM1 cargo-binding pocket, abrogating most, but not all, export functions of CRM1. KPT-0127 exerts a potent (EC50 300-400nM) and prolonged inhibition of CRM1-mediated HIV-1 Rev, forkhead (FOXO), and p53 nuclear export in a variety of normal and transformed cell lines. In cytotoxicity assays, KPT-0127 showed high potency in most hematologic cancer cell lines (EC50 <500nM, with leukemia and lymphoma lines often <100nM) and variable activity in solid tumor cell lines (EC50 90-2000nM), with colon and melanoma cells being the most sensitive (EC50< 100 nM). By contrast, normal cells were largely unaffected by treatment with KPT-0127 (EC50 >5-10µM). Studies in the HCT-116 colon cancer cell line suggested that KPT-0127 dose dependently increases the nuclear levels of p53 followed by an increase in caspase 3 activity, and appears to induce cell cycle arrest at both the G1/S and G2/M checkpoints, prior to inducing apoptosis. In normal peripheral blood mononuclear cells (PBMCs) and in Hut78 leukemia cells, KPT-0127 potently increased the nuclear levels of IB. However, KPT-0127 induced cell death of Hut78 cells with no effect on normal PBMCs. In drug combination studies, KPT-0127 showed additive or synergistic cytotoxicity activity with either 5-FU, carboplatin, or doxorubicin. Mechanism of action studies demonstrated that the Cys-528 residue in the cargo-binding pocket of CRM1 is essential for the inhibitory effect of KPT-0127. Mutagenesis of Cys-528 to a Ser completely abrogated KPT-0127 inhibition. KPT-0128, the trans isomer of KPT-0127, shows little effect on both HIV-Rev nuclear export and in cytotoxicity assays (EC50s > 10 µM), supporting the specificity of KPT-0127 for CRM1. Moreover, the selectivity of KTP-0127 was demonstrated across a panel of 37 proteins including several cysteine proteases.

In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (oral or SC) up to 560 mg/kg, and no deaths were observed. SC dosing daily for 5 days up to 100mg/kg (the highest dose tested) showed no behavioral, clinical chemistry, or hematogical effects in mice; Pharmacokinetics of KPT-0127 is adequate and in vivo efficacy studies are currently being performed; results will be presented. All together, these data demonstrate that KPT-0127 represents a novel, tumor selective and well-tolerated irreversible Crm1 inhibitor which may be suitable for clinical development both as a single agent and in combination with standard therapies.
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