Patient-derived Stem Research Articles

Therapeutic Targeting of Raf-Induced Paradoxical Erk Activation with a Vertical Combination Hitting Multiple Steps Along the Mapk Cascade

ABSTRACT Aim: Mounting evidence suggests that RAF-mediated MEK activation plays a crucial role in paradox MAPK (re)activation, leading to resistance and therapeutic failure with agents hitting a single step along the MAPK cascade. Methods: We examined the molecular and functional effects of single and combined MEK (trametinib, T), BRAF(dabrafenib, D), and pan-RAF (RAF265, R) inhibition, using WB and conservative isobologram analysis to assess functional synergism and explored genetic determinants of synergistic interactions. Results: In BRAF-mut models, both D and T effectively inactivated MAPK and inhibited cell growth, with no synergistic growth inhibition with their combination (CI: 0.7-1.3x106). Conversely, in KRAS-mut lung (2/5 cell lines) and pancreatic (4/6 cell lines) cancer models, in which selective BRAF inhibition induced paradox hyperphosphorylation of MEK, ERK, and p90RSK, combined D+T synergistically suppressed malignant growth (CI: 0.1-0.7). At concentrations inhibiting all RAF isoforms, R did not induce paradox MAPK activation and did not result in growth inhibitory synergism when combined withT. Highly synergistic in vitro growth inhibition was also observed with D+T in 2/5 patient-derived lung cancer stem cells. KRAS mutations appeared to be necessary, but not sufficient, to determine paradox MAPK activation and functional synergism with D+T, as assessed in two KRAS isogenic cell lines (H1299 expressing individual codon 12 mutants and HCT116 clones differing for homo or heterozygous G13D). Conversely, in lung cancer models driven by EGFR family activation (EGFR-mut HCC827 and H1650 cells and HER-2 overexpressing Calu-3 cells), paradox MAPK activation upon selective BRAF inhibition was dependent on upstream receptor activity and could be efficiently blocked by the EGFR/HER-2 inhibitor Lapatinib. Conclusions: Overall, our data indicate that RAF-mediated, paradox MAPK activation may be sustained by both upstream (RTK activation) and downstream (KRAS mutations) mechanisms and that in appropriate cellular contexts vertical RAF/MEK inhibition-based combination strategies may exert highly synergistic antitumor effects. Disclosure: All authors have declared no conflicts of interest.

MOLECULAR MOTORS AS NOVEL TARGETS TO BLOCK GLIOMA DISPERSION AND PROLIFERATION

BACKGROUND: Two of the defining features of glioblastoma are their ability to invade brain and to proliferate. While both processes are stimulated by the multiple signaling cascades that are activated in GBM, the redundancy present in these pathways has limited the clinical efficacy of kinase inhibitors. An alternative would be to directly target the cellular machinery that drives dispersion and proliferation. This machinery includes “molecular motors”—a superfamily of enzymes that generate the forces needed for a wide variety of cellular physiologies. In this study, we have examined two members from this family that can be targeted with clinically available drugs—cytoplasmic myosin II, which drives cellular motility, and Eg5, which drives the separation of the chromosomes during mitosis. METHODS: Rodent proneural glioblastomas were generated by intracranial injection of PDGF-expressing bi-cistronic retroviruses, and tumor cell invasion in vitro and ex vivo was examined as described (Ivkovic et al., Mol. Biol. Cell, 23, 533-42; Isermann et al., Curr. Protoc. Cell Biol., 2012). Patient-derived human glioma stem cells (GSCs) were isolated and maintained as described (Cheng et al., Cell, 153, 139-52). RESULTS: Myosin II: Myosin II isoforms are upregulated in all four subtypes of GBM, and expression correlates inversely with survival. We find that PDGF abolishes the anti-invasive effect of the EGFR inhibitior erlotinib on EGF-stimulated tumor dispersion, and, likewise, EGF abolishes the corresponding effect of the PDGFRα inhibitor imatinib on PDGF-stimulated tumor migration. However, blebbistatin, an allosteric inhibitor of myosin II, effectively blocks glioma dispersion even with simultaneous activation of both pathways. Furthermore, the rho kinase inhibitor fasudil, which inhibits myosin II and is used clinically for the treatment of pulmonary hypertension, also blocks PDGF-stimulated tumor invasion. Eg5: We find that Eg5 is consistently overexpressed in GSCs, compared to non-GSCs and that GSCs exhibit a greater sensitivity to ispinesib, a clinically available Eg5 inhibitor, compared to non-GSCs. Eg5 inhibition compromises the tumor initiation capability of GSCs as well as tumor progression in an orthotopic xenograft model. CONCLUSIONS: Prior efforts to block glioma invasion and proliferation have been hampered both by therapy-resistant GSCs and the redundancy present in pro-migratory signaling cascades. Our results indicate that molecular motors represent a set of targets whose inhibition blocks glioma dispersion and proliferation regardless of the activity of upstream growth and dispersion-promoting pathways and they imply that further pre-clinical and clinical development of molecular motor inhibitors in malignant glioma is warranted. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

822: The histone acetyltransferases inhibitor CPTH6 preferentially inhibits proliferation of patient-derived lung cancer stem cells in vitro and in vivo

822: The histone acetyltransferases inhibitor CPTH6 preferentially inhibits proliferation of patient-derived lung cancer stem cells in vitro and in vivo

Abstract 252: Molecular characterization of cancer stem cells isolated from primary colon cancer cells.

Abstract Colon cancer is the 3rd most common type of cancer and the 4th leading cause of cancer-related deaths worldwide. OncoTrack is an international consortium that has launched one of Europe's largest collaborative academic-industry research projects to develop and assess novel approaches for identification of new markers for colon cancer (1). To this end, colorectal tumors and metastases, as well as isolated circulating tumor cells and cancer stem cells are subjected to high-throughput sequencing of DNA and RNA. The laboratory data are combined with clinical data and used for in silico modeling of the patients' tumors and prediction of biomarkers as well as a tailored therapy. These predictions are validated using patient-specific in vitro (primary cells) and in vivo (xenografts) models. Recent data show that a small subpopulation of cells within different tumors, including colon cancer, is responsible for tumor growth and maintenance. Due to their functional similarities to adult tissue stem cells and their likely origin, these cells have been termed cancer stem cells. We aim at characterizing patient-derived primary cell cultures and cancer stem cells isolated from these. Primary tumors and metastases from colon cancer patients are cultivated in vitro using a matrigel-based, serum-free culture system. This system allows for long-term expansion of primary colon cancer cells in vitro (2). Using this methodology, we were able to establish over 35 novel, patient-specific colon cancer cell lines, both from primary tumors and metastases. Using previously established markers of colon cancer stem cells (CD44+, CD133+, CD166+, EpCAM-high, Wnt-high), cells are sorted by flow cytometry and subsequently subjected to genome-wide analyses using next-generation sequencing. These experiments allow for a detailed characterization of colon cancer stem cells at the level of transcriptome and methylome. It has been shown that tumors are heterogeneous with regard to gene mutations and expression of cancer stem cell markers (3). Using molecular tools such as padlock probes (4), we are analyzing the distribution of gene mutations identified by next-generation sequencing within primary cells as well as primary tumors. Moreover, we are addressing the expression pattern of cancer stem cell markers in these cell types. These data will help in a better understanding of individual tumors and assist in design of a personalized therapy. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). 1 Elsner M, Nature Biotechnology 29, 378 (2011); www.oncotrack.eu 2 Sato T el al., Gastroenterology. 2011 Nov;141(5):1762-72. 3 Gerlinger M et al., N Engl J Med. 2012 Mar 8;366(10):883-92. 4 Larsson C, et al., Nat Methods. 2010 May;7(5):395-7. Citation Format: Martin Lange, Dirk Schumacher, Thomas Hauling, Christian Regenbrecht, Oliver Politz, Mats Nilsson, Jens Hoffmann, Reinhold Schaefer, David Henderson. Molecular characterization of cancer stem cells isolated from primary colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2013-252

Use of PTEN status to determine the functional outcome of combined MEK and mTOR inhibition.

e13512 Background: PTEN is a lipid phosphatase couteracting the activity of the PI3K pathway. PTEN mutations and deficiencies are prevalent in many types of human cancers and are associated with poor prognosis and therapeutic resistance. MAPK is another key cellular network that works independently from, in parallel to, and/or through interconnections with PI3K to promote cancer development. Here we investigated whether a strategy combining MEK and mTOR inhibition may be effective in preclinical models of human cancer and the role of PTEN loss in determining sensivity/resistance to single and combined pathway inhibition. Methods: We employed in vitro assays (cell proliferation assays, cell cycle and apoptosis analysis, WB, and ELISA assays) to determine functional and molecular drug effects. Pharmacologic interactions between MEK and mTOR inhibitors were analyzed by conservative isobologram analysis using a fixed dose-ratio experimental design. Results: In cell lines with wt-PTEN (including melanoma, breast, lung, and colon cancer), combined MEK and mTOR blockade achieves synergistic effects at suboptimal drug concentrations but becomes frankly antagonistic in the presence of complete inhibition of MEK-to-ERK signaling (combination index - CI: 1.2->1000). This, in turn, led to the identification of a novel crosstalk mechanism by which MEK blockade restores PTEN expression and cross-inhibits the PI3K/AKT/mTOR pathway, thus bypassing the need for double pathway blockade. Conversely, in cancer cell lines with PTEN loss, combined MEK and mTOR blockade resulted in strongly synergistic effects (CI: 0.0005-0.4), due to cooperative induction of apoptosis. A similar synergistic growth inhibition was also observed in a patient-derived lung cancer stem cell line, which displayed low to undetectable PTEN protein levels. Conclusions: Our results suggest that combined MEK and mTOR inhibition exerts strongly synergistic effects in cancer models with PTEN loss. This notion may be helpful in selecting appropriate cellular (and possibly clinical) contexts for the design rational therapeutic strategies based on concomitant inhibition of the RAF/MEK/ERK and PI3K/AKT/mTOR pathways.

Abstract LB-466: Genomic Loss of microRNA 491 contributes to IGFBP2 overexpression and stemness in GBM

Abstract Insulin-like growth factor-binding protein 2 (IGFBP2) is overexpressed in 80% of glioblastoma multiforme (GBM, WHO grade IV). Our mouse model experiments demonstrated that IGFBP2 is a glioma oncogene. However, the underlying mechanism for overexpression of IGFBP2 remains unknown. We examined a total of 388 GBM samples from the Cancer Genome Atlas (TCGA) and found that miR-491, which is located on chr9p21.3, is deleted in 57% of cases. TargetScan analysis revealed that there is a binding site of miR-491 on the 3′-UTR of the IGFBP2 gene. The miR-491 expression levels in GBM patients are inversely correlated with that of IGFBP2. We validated this regulatory relationship by overexpression of miR-491 and observed down-regulation of IGFBP-2 in both protein level and mRNA level in U251 and T98G glioma cell lines. Conversely, miR-491 inhibitors increased IGFBP2 in LN229 cells which express low level of endogenous IGFBP2. Luciferase reporter assay further proved that IGFBP2 is a direct target of miR-491. Moreover, ectopic expression of miR-491 inhibited cell migration and invasion of U251 and T98G as efficiently as knockdown of IGFBP2 and such inhibitory effects can be partially reversed by overexpression of IGFBP2-EGFP in U251, supporting the notion that IGFBP2 plays an important role in miR-491's functions. To investigate the role of miR-491 in stemness of GBM, we introduced miR-491 into 3 patient-derived glioma stem cell lines and found that miR-49 downregulated CDK6 and Bcl-xL and inhibited neurosphere formation and cell proliferation. These findings suggest that miR-491 acts as a tumor suppressor miR by coordinately attenuating multiple oncogenes in GBM. Our results provide new insights into the mechanism for oncogenic IGFBP2 overexpression and may provide new opportunities for therapeutic strategies for GBM treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-466. doi:1538-7445.AM2012-LB-466

Abstract 5203: Lineage-specific mechanisms regulating PDGFRα oncogenic activity in glioblastoma

Abstract The highly heterogeneous nature of human tumors is a confounding feature which hinders understanding of the biology of cancer as well as design of effective therapies. Although a number of oncogenes and tumor suppressors, such as PI3K, Ras, TP53, CDKN2A, are frequently altrered across cancers of different origin, it is now apparent that cell lineage exerts a substantial effect on the distribution of genetic alterations in tumors. Consistent with the more recent notion that normal tissue stem cells or partially differentiated progenitor cells may serve as the cells of origin that undergo oncogenic transformation, it is conceivable to think that the cellular programs governing normal lineage development do also underlie the tumorigenic mechanisms. Therefore, the recognition of the tumor dependencies to oncogene functions in a lineage-specific context may offer more meaningful approaches for targeted cancer therapy. PDGF/PDGFRA signaling plays a particularly important role in the development of the brain, as a potent mitogen of oligodendrocyte precursor cells; it is also expressed in the type B neural stem cell population in the subventricular zone of adult brain. PDGF signaling has also been widely implicated in the formation of brain tumors. The Cancer Genome Atlas effort has revealed that overexpression and/or amplification of PDGFRA is the key feature of the proneural subtype of Glioblastomas (GBM), which constitutes 20-30% of all GBMs, occurs in younger patients and is extremely resistant to chemo- and radio-therapy. In proneural GBMs, PDGFRA expression clusters together with other proneural developmental genes such as Olig2 and Sox2. The targeting of PDGFRα has shown limited success in the setting of randomized clinical trials and in in vitro studies employing stable tumor cell lines. However, using a clinically relevant model, our data show that fresh patient-derived GBM stem like cells (Pt-GSLCs) belonging to the proneural subtype are particularly vulnerable to the inhibition of PDGFRα, both in vitro and in vivo, indicating a possible state of addiction to PDGFRα signaling. Interestingly, we observed that the neural transcription factor Olig2, might play a role in PDGFRα addiction. Inhibition of PDGFRα decreased phosphorylation of Olig2, while PDGFRα-inhibitor resistant lines upregulate the levels of Olig2 protein. Moreover Olig2 inhibition potentiates the effects of PDGFRα inhibition in proneural GBMs. Finally, PDGFRα and Olig2 inhibitors sensitize proneural GBM to radiation. These results provide an indication that oncogene deregulation may be “conditioned” by tumor lineage. We are now further investigating the lineage-specific mechanisms regulating addiction to PDGFRα of the proneural subtype of GBM with the hope to establish new pharmacological targets for the treatment of Glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5203. doi:1538-7445.AM2012-5203

Abstract 5118: A functional genetic approach in patient-derived glioblastoma stem cells reveals pre-mRNA splicing components to be cancer-lethal gene targets

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults and is among the deadliest cancers with a median survival period of 12-14 months. This poor prognosis despite aggressive therapy underscores the need for novel therapeutic targets specifically required by GBM cells. Many GBM are thought to arise from a neural stem cell (NSC) origin and, consistent with this premise, tumor-initiating GBM stem cells (GSCs) isolated from patients retain the NSC-like phenotype and molecular profile of primary tumors. Importantly, unlike serum-cultured cell lines, GSCs retain the developmental potential and specific genetic mutations acquired as each patient's tumor progressed from its cell of origin. We hypothesized that these genetic alterations driving GBM growth might also give rise to unique molecular vulnerabilities within the cancer cells. To identify such novel gene targets required for GBM cell growth, but which are dispensable to normal cells, we performed genome-scale RNAi screens in multiple patient-derived GSC isolates and simultaneously counter-screened against primary untransformed human NSCs. From these results, we identified and validated the existence of GBM-lethal genes that, when inhibited, render patient GSCs sensitive to cellular stresses arising within these transformed cells. From these targets, we show that GSCs have an increased requirement for the expression and function of multiple specific members of the pre-mRNA splicing machinery. Notably, loss of specific key splicing proteins resulted in cell cycle arrest and subsequent cell death only in GSCs, identifying the spliceosome as a specific molecular vulnerability in GBM. New treatment strategies for this disease are urgently needed. The identification of spliceosomal proteins as essential for the growth and maintenance of GSCs both adds to our understanding of glioblastoma biology and suggests novel targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5118. doi:1538-7445.AM2012-5118

Overview of the Current Issues and Advances in Haemopoietic Stem Cell Transplantation for β-thalassemia Major

Bone marrow transplantation (BMT) is the only possible curative treatment for β-thalassemia major. The largest experience occurred in Pesaro, Italy, where the BMT was applied after a standard risk assessment. The patients were divided into 3 risk classes based on liver size by physical examination, the presence or absence of fibrosis by liver biopsy, and adherence to regular iron chelation. Outcomes were mainly affected by the risk status. After modifications to the conditioning regimens, the risk of transplantation-related complications in highrisk recipients reduced considerably. As a result, outcomes after transplantation have become more similar across risk categories. For BMT, most centers use bone marrow instead of peripheral blood in thalassemia. Some studies showed that peripheral blood stem cell transplantation (PBSCT) is better than BMT with regard to hematologic recovery, hospitalization period, leukemia-free survival, overall survival (OS), and transplant-related mortality (TRM). No significant differences were seen in grade II to IV acute GVHD (aGVHD); but the incidence of chronic GVHD (cGVHD) was significantly higher in the PBSCT group. BMT from unrelated donors may offer similar results to those obtained using HLA-identical family donors, at least for patients who are not fully compliant with conventional treatment and do not yet show severe complications of iron overload. All studies conclude that MUD BMT might be a good alternative for patients with less risk factors. Another study concluded that, at present, due to high graft failure and GVHD rates, BMT from alternative donors should be restricted to patients who have poor life expectancies because they cannot receive adequate conventional treatment or because of alloimmunization to minor blood antigens. In another study unrelated cord blood transplantation (CBT) was compared to related donor transplantation for children with β-thalassemia. The results were comparable to the survival rates of related-donor BMT for thalassemia. It has always been a dream for parents to have a new baby who might be a donor for his/her sibling and save his/her life. Today some families tried to learn the HLA group of the fetus using prenatal diagnosis. The last step in this development was preimplantation genetic diagnosis (PGD). PGD has become available as an alternative to prenatal diagnosis in order to avoid the risk for pregnancy termination, because PGD allows selection of unaffected embryos before a pregnancy is established. Gene therapies, the ultimate idea, involves replacing allogeneic stem cell transplantation with the transfer of normal globin genes into patientderived autologous haematopoietic stem cells, bypassing the need for allogeneic donors and the immunosuppression required to achieve engraftment of the transplanted cells and to eliminate the risk of donor-related graft-versus-host disease. The successful preclinical studies in thalassaemia mousemodels, the accumulating data on lentiviral vector-mediated HSC transduction and the anticipated increased safety of lineage-restricted globin SIN lentiviral vectors strongly support the initiation of Phase I gene therapy clinical trials in β-thalassaemia.

Abstract 5033: Stromal-derived conditioned media activates STAT3 via JAK2 and TYK2 and results in de novo resistance to Nilotinib

Abstract Complete eradication of CML has not been possible in patients because of the presence of minimal residual disease (MRD) in sanctuary sites like the bone marrow (BM). One of the reasons for MRD is the milieu of cytokines and growth factors present in the BM microenvironment, that contribute to BCR-ABL independent drug resistance in CML. For example, in the present study, we demonstrate the importance of STAT3-mediated drug resistance in the BM microenvironment by showing that conditioned media (CM) derived from both normal and patient-derived mesenchymal stem cells and osteoblasts leads to STAT3 phosphorylation (pSTAT3-Y705) and concomitant drug resistance to Nilotinib in CML cells. Inhibiting the activity or reducing the expression of JAK2 and TYK2 was necessary for sensitizing cells to Nilotinib in CM. Moreover, increased pSTAT3-Y705 levels were associated with increased STAT3 transcriptional activity and up-regulation of Mcl-1 and cyclin D1. Our study indicates that there is an inherent redundancy in CM-mediated STAT3 activation pathway because concomitant neutralization of two potent activators of STAT3, IL-6 and G-CSF, does not reverse CM-mediated protection against Nilotinib. Furthermore, utilizing a SCID-hu bone implant in vivo model, we show that K562 cells with reduced STAT3 showed significantly lower tumor burden, which could be attributed to not only the slower proliferation rate, but also to the observation that these cells underwent cell death in BM stromal derived CM. Finally, CD34+ cells from CML patients when exposed to CM showed an increase in STAT3 phosphorylation which correlated with the development of de novo resistance to Nilotinib-mediated cell death. Taken together our data indicate that STAT3 is an important target in CML and its direct inhibition, along with BCR-ABL inhibitors, will likely be the most effective therapeutic strategy for reversing resistance within the BM microenvironment. 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 5033. doi:10.1158/1538-7445.AM2011-5033

Abstract 4483: Establishing patient-derived colorectal cancer stem cell models with a PIK3CA mutation for the development of inhibitory drugs as targeted therapies

Abstract Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.