Search

Supplementary Figures 1-4 from Small Molecule Targeting the Hec1/Nek2 Mitotic Pathway Suppresses Tumor Cell Growth in Culture and in Animal

The response to immunotherapy could be better predicted by using a wide set of biomarkers, including serum tumor markers; however, robust immune markers associated with efficacy have yet to be validated. In this study, changes in immune cell subsets from NSCLC patients treated with anti-PD1 therapy were longitudinally monitored by high-dimensional cytometry by time of flight (CyTOF). The frequencies of circulating CD8+ and CD8+CD101hiTIM3+ (CCT T) subsets were significantly correlated with clinical response and survival. Enrichment of these populations in peripheral blood mononuclear cells (PBMCs) indicated a poor clinical response to ICB therapy. Cell function assays revealed that these subsets were remarkably impaired, which supported the poor outcomes observed. Additionally, longitudinal analysis showed that KLRG1 expression and cytokines were associated with the response to therapy. Overall, our results provide novel potential biomarkers for guiding the management of NSCLC patients eligible to anti-PD-1 therapy, and contribute insights for new therapeutic strategies. Funding: The research leading to these results has received funding from the Macau Science and Technology Development Fund (Project no: 0096/2018/A3 & 001/2020/ALC), NSFC overseas and Hong Kong and Macao scholars cooperative research fund project (Project no: 81828013) and Janssen therapeutic fund (Project code: ICD#1101175) as well as The 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund (Guangdong- Hong Kong-Macau Joint Lab) (Project no: 2020B1212030006). Declaration of Interest: None to declare. Ethical Approval: This study was approved by Kiang Wu Hospital under the approval number 2018-007.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:12 pages:2317-2328 ispartof: location:United States status: published

RAD51 recombinase activity plays a critical role for cancer cell proliferation and survival, and often contributes to drug-resistance. Abnormally elevated RAD51 function and hyperactive homologous recombination (HR) rates have been found in a panel of cancers, including breast cancer and chronic myeloid leukaemia (CML). Directly targeting RAD51 and attenuating the deregulated RAD51 activity has therefore been proposed as an alternative and supplementary strategy for cancer treatment. Here we show that a newly identified small molecule, IBR2, disrupts RAD51 multimerization, accelerates proteasome-mediated RAD51 protein degradation, reduces ionizing radiation-induced RAD51 foci formation, impairs HR, inhibits cancer cell growth and induces apoptosis. In a murine imatinib-resistant CML model bearing the T315I Bcr-abl mutation, IBR2, but not imatinib, significantly prolonged animal survival. Moreover, IBR2 effectively inhibits the proliferation of CD34(+) progenitor cells from CML patients resistant to known BCR-ABL inhibitors. Therefore, small molecule inhibitors of RAD51 may suggest a novel class of broad-spectrum therapeutics for difficult-to-treat cancers.

Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38. In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and 'Sensitive' tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL. FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either. This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC. To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment. Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. Disclosures Zhou: Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

High expression in cancer 1 (Hec1) is an oncogene overly expressed in many human cancers. Small molecule inhibitor of Nek2/Hec1 (INH) targeting the Hec1 and its regulator, Nek2, in the mitotic pathway, was identified to inactivate Hec1/Nek2 function mediated by protein degradation that subsequently leads to chromosome mis-segregation and cell death. To further improve the efficacy of INH, a series of INH analogues were designed, synthesized, and evaluated. Among these 33 newly synthesized analogues, three of them, 6, 13, and 21, have 6-8 fold more potent cell killing activity than the previous lead compound INH1. Compounds 6 and 21 were chosen for analyzing the underlying action mechanism. They target directly the Hec1/Nek2 pathway and cause chromosome mis-alignment as well as cell death, a mechanism similar to that of INH1. This initial exploration of structural/functional relationship of INH may advance the progress for developing clinically applicable INH analogue.

Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI50, 10–21 μmol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds. [Cancer Res 2008;68(20):8393–9]

The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates.

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.

As cells differentiate into tissues, the microenvironment that surrounds these cells must cooperate so that properly organized, growth-controlled tissues are developed and maintained. We asked whether substances produced from this collaboration might thwart malignant cells if they arise in the vicinity of normal tissues. Here, we identified six factors secreted by nonmalignant mammary epithelial cells (MECs) differentiating in three-dimensional laminin-rich gels that exert cytotoxic activity on breast cancer cells. Among these, interleukin-25 (IL-25/IL-17E) had the highest anticancer activity without affecting nonmalignant MECs. Apoptotic activity of IL-25 was mediated by differential expression of its receptor, IL-25R, which was expressed in high amounts in tumors from patients with poor prognoses but was low in nonmalignant breast tissue. In response to IL-25, the IL-25R on the surface of breast cancer cells activated caspase-mediated apoptosis. Thus, the IL-25/IL-25R signaling pathway may serve as a new therapeutic target for advanced breast cancer.

Chromokinesins are microtubule-motor molecules that possess chromatin binding activity and are important for mitotic and meiotic regulation. The chromokinesin-member Kif4A is unique in that it localizes to nucleus during interphase of the cell cycle. Kif4 deletion by gene targeting in mouse embryonic cells was known to associate with DNA damage response. However, its precise role in DNA damage or repair pathway is not clear. Here we report that Kif4A associates with BRCA2 in a biochemical identification and that the interaction is mediated by the Kif4A C-terminal cargo-binding domain and BRCA2 C-terminal conserved region. Upon nucleus-specific laser micro-irradiation, Kif4A was rapidly recruited to sites of DNA damage. Significantly, the depletion of Kif4A from cells by shRNA impaired the ionizing-radiation induced foci (IRIF) formation of Rad51, both quantitatively and qualitatively. In contrast, the IRIF of gamma-H2AX or NBS1 was largely intact. Moreover, Kif4A knockdown rendered cells hypersensitive to ionizing radiation in a colonogenic survival assay. We further demonstrated that Kif4A deficiency led to significantly decreased homologous recombination in an I-SceI endonuclease induced in vivo recombination assay. Together, our results suggest a novel role for a chromokinesin family member in the DNA damage response by modulating the BRCA2/Rad51 pathway.

Three-dimensional explant cultures of muscle tissue were used to characterize secreted proteins regulated by endogenous levels of the angiogenesis modulator thrombospondin (TSP)-1. Explants from TSP1 null mice exhibit enhanced neovascularization associated with increased endothelial outgrowth but decreased outgrowth of perivascular smooth muscle cells . The absence of endogenous TSP1 did not diminish activation of latent transforming growth factor-beta and moderately decreased matrix metalloproteinase levels. However, significant changes in other secreted proteins were observed. Endogenous TSP1 decreased mRNA levels for collagens Ialpha1, Ialpha2, and IIIalpha1 and laminin alpha4 and increased collagen IValpha1 mRNA expression. Endogenous TSP1 also decreased the level of type I collagen protein produced by the vascular outgrowths. Collagens Ialpha1, Ialpha2, and IIIalpha1 are known tumor endothelial markers, suggesting that TSP1 coordinately regulates a set of extracellular matrix genes that reverse the angiogenic switch. Suppression of collagen Ialpha1 or Ialpha2 mRNAs using antisense morpholinos inhibited outgrowth in TSP1 null explants and proliferation of TSP1 null endothelial cells, indicating that type I collagen synthesis is limiting for this neovascularization response.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.

We examined the function of α4β1integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. α4β1supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by α4β1, whereas antagonism of fibroblast growth factor-2–stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an α4β1-dependent manner. Soluble α4β1antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1–dependent and not observed in explants from thrombospondin-1−/−mice. Antagonizing α4β1may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the α4β1binding sequence stimulate angiogenesis in vivo. Therefore, α4β1is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.

Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is predictive of poor patient overall survival in a variety of human cancers including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). Axl expression is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells and Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, stem cell-like traits and resistance to targeted inhibitors and other chemotherapeutic agents. BGB324/R428 is an oral, selective small molecule inhibitor of Axl that recently was evaluated in early clinical safety studies in healthy volunteers. Treatment with BGB324 up to and including 1.5 gms daily (per os) was established as being safe and well tolerated. The endpoints of the study included pharmacokinetics and safety. Bioavailability was increased in the presence of food and systemic exposure increased dose proportionately. At the highest dose the apparent elimination half-life approached four days presenting a range of dosing options. We evaluated the effects of BGB324 in preclinical models of TNBC, PDA and NSCLC, including 2D/3D cell culture and mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 treatment of mesenchymal carcinoma cells blocked invasiveness and enhanced chemotherapeutic efficacy. BGB324 abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells. BGB324 treatment blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDA. Collectively, these data suggest that the first-in-class selective Axl inhibitor BGB324 can overcome EMT-related acquired therapeutic resistance and enhance the efficacy of multiple anti-cancer strategies. Together with the results of results of the early clinical safety studies, this provides a rationale for further clinical studies. Citation Format: Katarzyna Wnuk-Lipinska, Crina Tiron, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blø, David Micklem, Murray Yule, John Minna, Longen Zhou, Rolf Brekken, James Lorens. BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1747. doi:10.1158/1538-7445.AM2014-1747

Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is associated with a variety of human cancers including non-small cell lung carcinoma (NSCLC), and is predictive of poor patient overall survival. Axl is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells. Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, and can confer resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), as well as other chemotherapeutic agents. BGB324 is a potent, reversible and selective small molecule inhibitor of Axl that has recently entered into phase I clinical trials. We evaluated the effects of BGB324 on NSCLC cells in in vitro 3D assays and in mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 in combination with EGFR TKI erlotinib demonstrated a synergistic anti-proliferation effect on NCI-H1299 (mesenchymal, EGFR wild-type, erlotinib-resistant) human NSCLC cells in 3D culture. In mouse xenograft models using NCI-H1299 cells BGB324 treatment significantly enhanced the antitumor acitivity of Docetaxel. Similarly, using the mesenchymal NSCLC cell line A549 (EGFR wild-type), the combination of BGB324 with either erlotinib or anti-VEGF agent bevacizumab showed synergistic or additive anti-tumour activity. Furthermore, in HCC827 (EGFR mutant, erlotinib-sensitive) human NSCLC xenograft model, addition of BGB324 treatment strikely delayed the emergence of acquired resistance to erlotinib. Taken together, these data suggest the first-in-class selective Axl inhibitor BGB324 can overcome acquired resistance in in vivo models of NSCLC. Studies in human NSCLC patient-derived xenograft models and mechanisms of action are in progress. Citation Format: Katarzyna Wnuk-Lipinska, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Hallvard Haugen, Liang Xiao, Magnus Blø, David Micklem, John Minna, Rolf Brekken, Longen Zhou, James Lorens. Selective small molecule AXL inhibitor BGB324 overcomes acquired drug resistance in non-small cell lung carcinoma models. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B30.

Rad51 recombinase is a key downstream effector of BCR-ABL, essential for DNA repair, cell proliferation and survival. In BCR-ABL positive leukemia cells, Rad51, itself a direct substrate of BCR-ABL, is transcriptionally activated through the BCR/ABL-Stat5 pathway. We have identified a novel compound IBR2, which targets Rad51 and inhibits its functions both in vitro and in vivo. We investigated whether targeted inhibition of Rad51 by IBR2 could overcome imatinib-resistance in CML. IBR2 potently inhibited the proliferation of Ba/F3 cells expressing wild-type BCR-ABL (P210), T315I or E255K mutant with the IC50 values (12–20 μM), but had much lesser effect on the Ba/F3 parental cells. By annexin-V staining and FACS analysis, treatment with 20 μM IBR2 for 48 hours significantly killed Ba/F3 cells expressing P210, E255K or T315I with 33.6, 35.8 and 66.5% of apoptotic cells, respectively. The effects of IBR2 on protein expression and phosphorylation of BCR-ABL and Stat5 and Rad51 were examined by Western blotting after 48 hours of exposure to IBR2 (10–20 μM). In Ba/F3 cells expressing BCR-ABL, dose-dependent decreases of Rad51, BCR-ABL, phosphorylated BCR-ABL, and phosphorylated Stat5 were revealed, while c-Abl, p-c-Abl and total Stat5 protein were unchanged. In contrast, there was little effect on Ba/F3 parental cells. In a murine imatinib-resistant chronic myelogenous leukemia (CML) model bearing T315I mutant BCR-ABL, IBR2 at the dose of 100mg/kg daily for 20 days (i.p.) significantly prolonged animal survival. By colony-forming cell (CFC) assays, IBR2 did not significantly inhibit the growth of cord blood progenitor cells (less than 10% up to 40 μM). However, IBR2 at concentrations of 25, 30 and 40 μM could effectively inhibit the growth of CD34+ progenitor cells from CML patients with demonstrated clinical resistance to imatinib and cross-resistance to Nilotinib by 38.9, 54.8 and 95.5%, respectively. In contrast, these cells were inhibited with 5 μM imatinib by 35.2%; with 5 μM nilotinib by less than 9%; and with 0.5 μM dasatinib by up to 41.3%. Synergistic effects were observed upon co-treatment of IBR2 with imatinib in Ba/F3 cells expressing wild-type BCR-ABL. Treatment with 15 μM IBR2 alone resulted in