Tumor Re-emergence Research Articles

Robust IL-2-dependent antitumor immunotherapy requires targeting the high-affinity IL-2R on tumor-specific CD8+ T cells

BackgroundDevelopment of interleukin (IL)-2-dependent antitumor responses focus on targeting the intermediate affinity IL-2R to stimulate memory-phenotypic CD8+ T and natural killer (NK) cells while minimizing regulatory T cell (Treg) expansion....

Blocking Gi/o-Coupled Signaling Eradicates Cancer Stem Cells and Sensitizes Breast Tumors to HER2-Targeted Therapies to Inhibit Tumor Relapse.

Simple SummaryCancer stem cells (CSCs) are associated with therapeutic resistance and tumor relapse but effective approaches for eliminating CSCs are still lacking. The aim of this study was to assess the role of G protein-coupled receptors (GPCRs) in regulating CSCs in breast cancer. We showed that a subgroup of GPCRs that coupled to Gi/o proteins (Gi/o-GPCRs) was required for maintaining the tumor-forming capability of CSCs in HER2+ breast cancer. Targeting Gi/o-GPCRs or their downstream PI3K/AKT and Src pathways was able to enhance HER2-targeted elimination of CSCs and therapeutic efficacy. These findings suggest that targeting Gi/o-GPCR signaling is an effective strategy for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor recurrence.Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block the tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.

Patient-Derived Bone Marrow Spheroids Reveal Leukemia-Initiating Cells Supported by Mesenchymal Hypoxic Niches in Pediatric B-ALL.

B-cell acute lymphoblastic leukemia (B-ALL) results from the expansion of malignant lymphoid precursors within the bone marrow (BM), where hematopoietic niches and microenvironmental signals provide leukemia-initiating cells (LICs) the conditions to survive, proliferate, initiate disease, and relapse. Normal and malignant lymphopoiesis are highly dependent on the BM microenvironment, particularly on CXCL12-abundant Reticular (CAR) cells, which provide a niche for maintenance of primitive cells. During B-ALL, leukemic cells hijack BM niches, creating a proinflammatory milieu incompetent to support normal hematopoiesis but favoring leukemic proliferation. Although the lack of a phenotypic stem cell hierarchy is apparent in B-ALL, LICs are a rare and quiescent population potentially responsible for chemoresistance and relapse. Here, we developed novel patient-derived leukemia spheroids (PDLS), an ex vivo avatar model, from mesenchymal stromal cells (MSCs) and primary B-ALL cells, to mimic specialized niche structures and cell-to-cell intercommunication promoting normal and malignant hematopoiesis in pediatric B-ALL. 3D MSC spheroids can recapitulate CAR niche-like hypoxic structures that produce high levels of CXCL10 and CXCL11. We found that PDLS were preferentially enriched with leukemia cells displaying functional properties of LICs, such as quiescence, low reactive oxygen species, drug resistance, high engraftment in immunodeficient mice, and long-term leukemogenesis. Moreover, the combination of PDLS and patient-derived xenografts confirmed a microenvironment-driven hierarchy in their leukemic potential. Importantly, transcriptional profiles of MSC derived from primary patient samples revealed two unique signatures (1), a CXCL12low inflammatory and leukemia expansion (ILE)-like niche, that likely supports leukemic burden, and (2) a CXCL11hi immune-suppressive and leukemia-initiating cell (SLIC)-like niche, where LICs are likely sustained. Interestingly, the CXCL11+ hypoxic zones were recapitulated within the PDLS that are capable of supporting LIC functions. Taken together, we have implemented a novel PDLS system that enriches and supports leukemia cells with stem cell features driven by CXCL11+ MSCs within hypoxic microenvironments capable of recapitulating key features, such as tumor reemergence after exposure to chemotherapy and tumor initiation. This system represents a unique opportunity for designing ex vivo personalized avatars for B-ALL patients to evaluate their own LIC pathobiology and drug sensitivity in the context of the tumor microenvironment.

Rapid screening for individualized chemotherapy optimization of colorectal cancer: A novel conditional reprogramming technology-based functional diagnostic assay.

BackgroundIn vitro patient tumor models such as patient-derived organoids (PDO) and conditionally reprogrammed (CR) cell culture are important for translational research and pre-clinical drug testing. In this study we present a personalized drug sensitivity test for late stage, potentially operable colorectal cancer (CRC) using patient-derived primary tumor cells isolated with i-CR technology, an optimized CR method. We explored the clinical feasibility of using i-CR platform to guide CRC chemotherapy, and established the correlation between in vitro drug sensitivity and patient clinical response. MethodsPrimary CRC tumor cells were isolated and cultured with the i-CR technology. NGS was performed and the WES and CNV results of i-CR cells were compared with that of the original patient tumor samples. In vitro drug screenings were done with guideline chemotherapy drugs for CRC. In vivo drug response was examined with paired PDX mouse models. A double-blind co-clinical cohort study was carried out and the clinical outcomes of the enrolled patients were compared with the i-CR results. Resultsi-CR platform could be used to rapidly propagate primary colorectal tumor cells that represent individual patient tumors effectively by keeping the clonal heterogeneity and the genetic characteristics. Chemotherapy drug screenings with i-CR cells were comparable with that of PDX models. More importantly, i-CR results showed high accordance with the clinical outcomes of the enrolled CRC patients. Conclusioni-CR platform was capable to test and optimize therapeutic regimens pre-clinically, study cancer cell biology, and model tumor re-emergence to identify new targeted therapeutics from an effective personalized medicine standpoint.

Abstract 3071: Repotrectinib demonstrates promising activities in ROS1 wild-type and solvent-front mutant lung cancer patients-derived preclinical models

Abstract The ROS1-rearranged non-small-cell lung cancer (NSCLC) is currently treated with ALK/ROS1 tyrosine kinase inhibitor (TKI) crizotinib as a first-line agent. However, resistance invariably develops and subsequent therapeutic option for overcoming them is limited. Therefore, we investigated the antitumor activity of several ROS1-TKIs in ROS1-positive patient-derived preclinical models. In this study, we established 6 patient-derived cells (PDCs) from patients with different types of ROS1 fusion partners as follows: treatment-naïve (YU1078, YU1082 and YU1083), crizotinib-resistant G2032R (YU1079) and crizotinib- or entrectinib- primary resistant (YU1081 and YU1085). Sanger sequencing and whole-exome sequencing were then conducted to determine the characteristics. The in vitro and in vivo antitumor activities of ROS1-TKIs in these PDCs were evaluated by comparing the survival, expression assay, duration of tumor re-emergence and ability to penetrate the blood-brain barrier (BBB). Repotrectinib potently inhibited cell proliferation and ROS1-downstream signaling pathways in YU1078 (CD74-ROS1). In YU1078-derived xenograft models, repotrectinib induced the marked tumor regression and delayed the duration of tumor re-emergence following drug withdrawal compared with crizotinib and entrectinib. Interestingly, both YU1081 (TPM3-ROS1) and YU1082 (SLC34A2-ROS1) with cross primary resistance to other clinically available ROS1-TKIs exhibited high sensitivity to repotrectinib in the concentration range of 100~200 nM. Immunoblot analysis demonstrated that repotrectinib completely suppressed the phosphorylation of ROS1 and STAT3. Notably, repotrectinib showed the selective and highly potency in vitro and in vivo against solvent-front mutant G2032R conferring crizotinib resistance. In G2032R-mutant xenograft models, the response of repotrectinib was still maintained until the end of the experiment (120 days) after drug withdrawal, but loratinib- or cabozantinib-treated tumors rapidly re-emerged. Moreover, repotrectinib induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration-time curve value. As clinical proof of concept, the potent systemic and intracranial activity of ropotrectinib has been observed in patients who had progressed on prior TKIs or TKI-naïve patients, who were enrolled on-going phase 1/2 clinical trial (NCT03093116). Our finding indicated a superior antitumor activity of repotrectinib in both wild-type and mutant ROS1 fusion, providing a rationale that repotrectinib may be an effective subsequent or upfront treatment option for patients with ROS1-positive NSCLC who have relapsed on the available TKIs as well as TKI naïve, including patients with progressive CNS metastases. Citation Format: Dong Hwi Kim, Seok-Young Kim, Hyeong Seok Joo, You Won Lee, Han Na Kang, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho, Miran Yun. Repotrectinib demonstrates promising activities in ROS1 wild-type and solvent-front mutant lung cancer patients-derived preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3071.

Abstract A33: Patient-specific evaluation of chemoresistance and tumor recurrence using ovarian cancer stem cell spheroids

Abstract Ovarian cancers grow in suspension in the ascites fluid, and contain a small population of ovarian cancer stem cells (OvCSC), which are resistant to therapy. Due to the rarity of OvCSCs, we developed a 3D hanging drop platform, in which as few as one ALDH+ CD133+ cell (isolated from primary malignant ascites) can be stably incorporated into 3D spheroids. Our platform can be utilized to quantify drug sensitivity of chemotherapeutic agents in the context of OvCSCs, distinguish drug responses for the same drugs between several patient samples, and model patient-specific tumor re-emergence, making it uniquely suited for the development of personalized therapeutics. Three patient samples (Pt259, Pt224, Pt152) were evaluated and robust proliferation rates were observed in spheroids, ranging from 5.3 fold to 8.4 fold. By Day 7, ALDH+ CD133+ cells had differentiated within spheroids to form progeny of ALDH- CD133-, ALDH+ CD133-, and CD133+ ALDH- cells while maintaining an ALDH+ CD133+ population. Each patient-derived spheroid demonstrated a different composition of these progeny, which were similar to those observed in the patient samples. OvCSC spheroids had differing responses to drug treatments (cisplatin, ALDH targeting compound 673A, and JAK1/2 inhibitor ruxolitinib). Combination of cisplatin/673A targeted ALDH+ and CD133+ in all patient samples. Pt259 samples were maximally sensitive to cisplatin/673A, while Pt224 and Pt152 were more resistant (20-40% higher viability). Combination dose of cisplatin/ruxolitinib targeted CD133+ populations. By isolating cells that escaped chemotherapy, we created a spheroid model to study tumor re-emergence. ALDH+ populations re-emerged to a lower extent compared to original OvCSC spheroids, while CD133+ populations did not recover at all. Spheroids formed from the most platinum-sensitive cells (Pt259) and the most platinum-resistant cells (Pt152) following cisplatin/673A treatment were also serially passaged over 7 cycles in 7 weeks to characterize CD133+ and ALDH+ populations and evaluate their ability to reform spheroids, effectively modeling tumor re-emergence in vitro. ALDH+ OvCSC progeny reliably repopulated within these spheroids despite initial depletion following treatment. Over six serial passages, ALDH+, CD133+, and ALDH+ CD133+ populations gradually returned to original and even higher than levels seen in original patient samples. Lastly, OvCSC spheroids initiated tumors in immunodeficient mice at 100% success with only 10 spheroids injected. These tumors demonstrated a distinct response to therapy that corresponds with responses seen in spheroids, indicating that our model may be a means to screen tumors for personalized drug selection. Our patient-derived low-cell-number OvCSC spheroid platform can be utilized to study tumor biology, to model tumor re-emergence after primary chemotherapy, and to identify new targeted therapeutics from a personalized medicine standpoint. Citation Format: Shreya Raghavan, Pooja Mehta, Michael Bregenzer, Maria Ward Rashidi, Elyse Fleck, Lijun Tan, Karen McLean, Ronald Buckanovich, Geeta Mehta. Patient-specific evaluation of chemoresistance and tumor recurrence using ovarian cancer stem cell spheroids. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A33.

Predictive bioinformatics identifies novel regulators of proliferation in a cancer stem cell model

The cancer stem cell model postulates that tumors are hierarchically organized with a minor population, the cancer stem cells, exhibiting unlimited proliferative potential. These cells give rise to the bulk of tumor cells, which retain a limited ability to divide. Without successful targeting of cancer stem cells, tumor reemergence after therapy is likely. However, identifying target pathways essential for cancer stem cell proliferation has been challenging. Here, using a transcriptional network analysis termed GAMMA, we identified 50 genes whose correlation patterns suggested involvement in cancer stem cell division. Using RNAi depletion, we found that 21 of these target genes showed preferential growth inhibition in a breast cancer stem cell model. More detailed initial analysis of 6 of these genes revealed 4 with clear roles in the fidelity of chromosome segregation. This study reveals the strong predictive potential of transcriptional network analysis in increasing the efficiency of successful identification of novel proliferation dependencies for cancer stem cells.

Highlights of This Issue

With the recent FDA-approval of pembrolizumab for mismatch repair (MMR)-deficient cancers, interest has emerged in identifying rapid and inexpensive methods to screen for MMR deficiency, especially in tumor types where this is uncommon. Guedes and colleagues employed an immunohistochemical assay to screen 1176 primary prostate cancers for loss of MSH2, the most commonly inactivated MMR protein in prostate cancer. While the prevalence of MSH2 deficiency was rare in the entire cohort (1%), MSH2 loss was enriched in patients with primary Gleason pattern 5 cancers (8%) and small-cell prostate cancers (5%). If validated, screening for MSH2 inactivation in high-risk patients could facilitate selection of patients for pembrolizumab.Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, an anti-angiogenic IgG monoclonal antibody. Bevacizumab prolongs survival but approximately 20% of patients show no anti-tumor response. Its ability to mitigate edema has further promoted its widespread use. In this study, Müller-Greven and colleagues demonstrate that anti-VEGF-A IgG is internalized by Sox2+/CD44+ glioblastoma stem-like tumor cells residing in the perivascular niche in patient-derived tumor and syngeneic models of GBM. Survival of CD133+ cancer stem-like cells is promoted by macropinocytosis of bevacizumab and its trafficking to the lysosome and by autophagy induced by bevacizumab depletion of VEGF-A within the microenvironment.Ovarian cancer is the most lethal gynecologic malignancy, with nearly 70% relapse within 24 months. Raghavan and colleagues have developed a patient-derived 3D hanging drop spheroid platform to screen chemotherapeutics and biologics and to improve clinical outcomes by rapidly identifying optimal personalized treatment routes. The authors show that spheroids formed with ovarian cancer stem cells from different patients exhibited distinct responses to chemotherapeutics, which corresponded with responses observed in primary tumor xenografts. Drug resistance was related to levels of stem cell markers CD133 and ALDH. This platform can be used to further study cancer stem cell biology and tumor re-emergence, leading to improved clinical outcomes.The ability to distinguish primary prostate cancer (PCa) with metastatic potential has not been achieved. Labbé and colleagues interrogated the impact of concurrent TOP2A and EZH2 expression in multiple PCa patient cohorts. Concurrent expression of both marks identified patients more susceptible to progress to metastatic disease and PCa-specific death. Importantly, TOP2A and EZH2 in PCa cells act as key driving oncogenes, highlighted by sensitivity to combination-targeted therapy. This work provides substantial evidence for the utility of TOP2A and EZH2 as prognostic biomarkers and therapeutic targets to identify and intercept aggressive PCa progressing to metastatic disease.

Personalized Medicine-Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids.

Purpose: Chemoresistant ovarian cancers grow in suspension within the ascites fluid. To screen the effect of chemotherapeutics and biologics on resistant ovarian cancers with a personalized basis, we developed a 3D hanging drop spheroid platform.Experimental Design: We initiated spheroids with primary aldehyde dehydrogenase-positive (ALDH+) CD133+ ovarian cancer stem cells (OvCSC) from different patient samples and demonstrated that stem cell progeny from harvested spheroids was similar to the primary tumor. OvCSC spheroids were utilized to initiate tumors in immunodeficient mice. Drug responses to cisplatin and ALDH-targeting compound or JAK2 inhibitor determined whether the OvCSC population within the spheroids could be targeted. Cells that escaped therapy were isolated and used to initiate new spheroids and model tumor reemergence in a personalized manner.Results: OvCSC spheroids from different patients exhibited varying and personalized responses to chemotherapeutics. Xenografts were established from OvCSC spheroids, even with a single spheroid. Distinct responses to therapy were observed in distinct primary tumor xenografts similar to those observed in spheroids. Spheroids resistant to cisplatin/ALDH inhibitor therapy had persistent, albeit lower ALDH expression and complete loss of CD133 expression, whereas those resistant to cisplatin/JAK2 inhibitor therapy were enriched for ALDH+ cells.Conclusions: Our 3D hanging drop suspension platform can be used to propagate primary OvCSCs that represent individual patient tumors effectively by differentiating in vitro and initiating tumors in mice. Therefore, our platform can be used to study cancer stem cell biology and model tumor reemergence to identify new targeted therapeutics from an effective personalized medicine standpoint. Clin Cancer Res; 23(22); 6934-45. ©2017 AACR.

Abstract AP22: CHEMORESISTANCE, TUMOR INITIATION AND TUMOR RE–EMERGENCE TRENDS IN MALIGNANT ASCITES–DERIVED OVARIAN CANCER STEM CELL SPHEROIDS

Abstract Due to the rarity of ovarian cancer stem cells (OvCSC; <0.2% total population), we developed a 3D hanging drop array model, in which as few as 10 OvCSCs (isolated from primary malignant ascites based on ALDH+ CD133+) can be stably incorporated into spheroids for study of drug sensitivity and tumor biology. Our platform can be utilized to: i) quantify drug sensitivity of chemotherapeutic agents in the context of OvCSCs; ii) distinguish drug responses for the same drugs between several patient samples; thereby uniquely suited for the development of personalized therapeutics including the context of OvCSCs. Three patient samples were evaluated (stage IIIcstage IV). Robust proliferation rates were observed in spheroids, ranging from 5.3 fold (progressive Pt.259++) to 6.5 fold(resistant Pt224++) to 8.4 fold(recurrent Pt152++). Examination of OvCSC populations by Day 7 indicated that ALDH+ CD133+ cells had differentiated within spheroids to form progenies of ALDH- CD133-, ALDH+ CD133-, and CD133+ ALDH- cells. Maintenance of the ALDH+ CD133+ pool was also observed (0.04-0.7%). Composition of progenies was different between the three different patient samples: In Pt259, <1% CD133+ and <5% ALDH+; Pt224 with ~25% ALDH+ progeny; and Pt152 with ~20% CD133+ progeny. OvCSC spheroids had differing responses to drug treatments (cisplatin, ALDH targeting compound 673A, and JAK1/2 inhibitor Ruxolitinib). Combination of cisplatin/673A targeted ALDH+ and CD133+ in all patient samples. Progressive Pt259 samples were maximally sensitive to Cisplatin/673A, while Pt224 and Pt152 were more resistant to drug(20-40% higher viability). Combination dose of Cisplatin/Ruxolitinib targeted CD133+ populations, but also resulted in increased ALDH+ cells, indicating a potential differentiation of CD133+ cells into ALDH+ cells to escape cell-death. By isolating cells that escape chemotherapy, we created a spheroid model to study tumor re-emergence. ALDH+ populations re-emerged to a lower extent compared to original OvCSC spheroids, while CD133+ populations did not recover at all. Lastly, OvCSC spheroids (10 cells/drop) initiated tumors in immunodeficient mice (2 spheroids – 80% success rate; 10 spheroids – 100% success rate). Conclusions: We have developed a hanging drop array model that incorporates primary ovarian cancer stem cells into a 3D spheroid, capable of initiating tumors in immuno-deficient mice. OvCSCs differentiate into heterogeneous progenies within spheroids, correlating with differing responses to conventional and stem cell targeting drug treatments, providing a platform for personalized therapeutics. We also developed a spheroid model that can mimic tumor re-emergence using cells that escape first line chemotherapy. This platform can be utilized to study spheroid and cancer stem cell biology, and model tumor re-emergence to identify new targeted therapeutics. This work was supported by the DOD OCRP Early Career Investigator Award W81XWH-13-1- 0134 (GM). Citation Format: Shreya Raghavan PhD, Maria Ward BS, Pooja Mehta MSc, Ronald J Buckanovich MD PhD, Geeta Mehta PhD. CHEMORESISTANCE, TUMOR INITIATION AND TUMOR RE–EMERGENCE TRENDS IN MALIGNANT ASCITES–DERIVED OVARIAN CANCER STEM CELL SPHEROIDS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP22.

A mouse model of MYCN-driven retinoblastoma reveals MYCN-independent tumor reemergence.

The most frequent focal alterations in human retinoblastoma are mutations in the tumor-suppressor gene retinoblastoma (RB) and amplification of the oncogene MYCN. Whether MYCN overexpression drives retinoblastoma has not been assessed in model systems. Here, we have shown that Rb inactivation collaborates strongly with MYCN overexpression and leads to retinoblastoma in mice. Overexpression of human MYCN in the context of Rb inactivation increased the expression of MYC-, E2F-, and ribosome-related gene sets, promoted excessive proliferation, and led to retinoblastoma with anaplastic changes. We then modeled responses to MYCN-directed therapy by suppressing MYCN expression in MYCN-driven retinoblastomas. Initially, MYCN suppression led to proliferation arrest and partial tumor regression with loss of anaplasia. However, over time, retinoblastomas reemerged, typically without reactivation of human MYCN or amplification of murine Mycn. A subset of returning retinoblastomas showed genomic amplification of a Mycn target gene encoding the miR cluster miR-17~92, while most retinoblastomas reemerged without clear genetic alterations in either Mycn or known Mycn targets. This Rb/MYCN model recapitulates key genetic driver alterations seen in human retinoblastoma and reveals the emergence of MYCN independence in an initially MYCN-driven tumor.

CEP-28122, a Highly Potent and Selective Orally Active Inhibitor of Anaplastic Lymphoma Kinase with Antitumor Activity in Experimental Models of Human Cancers

Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice.

Abstract 3574: Identification and preclinical characterization of CEP-28122, a highly potent and selective orally active inhibitor of anaplastic lymphoma kinase, in lymphoma and non-small cell lung cancer models

Abstract Anaplastic lymphoma kinase (ALK) was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas (ALCL). Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein-like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity (IC50 of 3 nM) and NPM-ALK tyrosine phosphorylation in ALCL cells (IC50 of 20-30 nM). It is selective over a broad panel of protein kinases and a panel of receptors and ion channels with greater than 300-fold selectivity for insulin receptor. CEP-28122 induced concentration-dependent growth inhibition and cytotoxicity of ALK-positive ALCL and NSCLC cells with minimal activity against ALK-negative lymphoma and leukemia cells as well as ALK-negative NSCLC cells at concentrations up to 3 μM. CEP-28122 exhibited favorable oral bioavailability (F = 37-71% across species) with adequate tissue distribution in rodents. It displayed dose-dependent inhibition of NPM-ALK tyrosine phosphorylation in human ALCL tumor xenografts in mice with complete target inhibition (> 90%) for more than 12 h following single oral dosing at 30 mg/kg. Dose-dependent anti-tumor activity was observed in NPM-ALK-positive Sup-M2 and Karpas-299 ALCL tumor xenografts and EML4-ALK-positive NCI-H2228 and NCI-H3122 tumor xenografts in mice dosed with CEP-28122, bid, po, with complete or near complete tumor regressions observed following 2 weeks of treatment with CEP-28122 at 30 mg/kg or higher. Treatment of mice bearing Sup-M2 tumor xenografts or primary human ALCL tumorgrafts with CEP-28122 at 55 or 100 mg/kg bid, po, for 4 weeks led to sustained tumor regression in all mice, with no tumor re-emergence in any mouse up to 60 days post cessation of CEP-28122 treatment. On the contrary, CEP-28122 displayed marginal anti-tumor activity against ALK-negative lymphoma and NSCLC tumor xenografts under the same dosing regimens. It was well tolerated with all dosing regimens in mice and rats with no overt toxicity and no compound-related body weight loss. CEP-28122 advanced into preclinical development based on its potency, selectivity and overall favorable pharmacological, pharmaceutical and safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2011-3574