Establishment of a t(11;19), KMT2A Rearranged B-ALL Cell Line for Preclinical Evaluation and Novel Therapeutics Development for Refractory Infant Leukemia.

Leukemia, diagnosed in children less than 12 months of age, is a rare condition with an aggressive disease presentation and poor response to conventional chemotherapeutic agents. In addition, the unique vulnerability of the affected population does not always permit the use of markedly intense regimens with higher doses of cytotoxic agents. However, the unique biology of these leukemic cells also provides opportunities for the identification of effective and potentially well-tolerated targeted therapeutic strategies. In this report, we describe the establishment and characterization of a cell line from the blasts of an infant diagnosed with refractory B-cell acute lymphoblastic leukemia (ALL) carrying the characteristic histone lysine methyltransferase 2A (KMT2A) gene rearrangement. This cell line consists of rapidly proliferating clones of cells with chemosensitivity patterns previously described for KMT2A rearranged leukemia cells, including relative resistance to glucocorticoids and sensitivity to cytarabine. We also show effective targetability with menin inhibitors, indicating the activity of abnormal KMT2A-related pathways and the potential utility of this cell line in comprehensive drug library screens. Overall, our findings report the establishment and in vitro validation of a cell line for research into key aspects of infant leukemia biology and targeted therapeutics development.

Targeted Polo-like Kinase Inhibition Combined With Aurora Kinase Inhibition in Pediatric Acute Leukemia Cells.

BACKGROUND: Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. MATERIALS AND METHODS: We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. RESULTS: Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. DISCUSSION: This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.

Polo-like kinase 1 inhibition kills glioblastoma multiforme brain tumor cells in part through loss of SOX2 and delays tumor progression in mice.

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo-like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1-high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110-470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G(2) /M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion.

BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

Ion channels in pediatric CNS Atypical Teratoid/Rhabdoid Tumor (AT/RT) cells: potential targets for novel therapeutic agents.

The central nervous system Atypical Teratoid/Rhabdoid Tumor (CNS AT/RT) is a highly malignant neoplasm that commonly affects infants and young children, and has an extremely poor prognosis. Recently, a small subset of ion channels have been found to be over-expressed in a variety of malignant cells, thus emerging as potential therapeutic targets for difficult to treat tumors. We have studied the electrophysiological properties of AT/RT cell lines with particular attention to cell volume sensitive ion channels (VSC). This class of membrane proteins can play a fundamental role in cellular processes relevant to tumor development. We have found that chloride selective VSCs are particularly active in AT/RT cell lines, compared to non-tumor cells. We evaluated specific inhibitors for activity against chloride selective VSCs and consequently for their ability to inhibit the growth and survival of AT/RT cells in vitro. The results demonstrated that the extent of volume sensitive membrane current inhibition by these agents was correlated with their potency in AT/RT cell growth inhibition in vitro. In addition, we showed that ion channel inhibition enhanced the activity of certain anti-neoplastic agents, suggesting its value in effective drug combination protocols. Results presented provide preliminary in vitro data for possible evaluation of distinct ion channels as plausible therapeutic targets in the treatment of AT/RT.

The T-type Calcium Channel Cav3.1 in Y79 Retinoblastoma Cells is Regulated by the Epidermal Growth Factor Receptor via the MAPK Signaling Pathway.

PURPOSE: Retinoblastoma is the most frequent intraocular cancer in children. It is also one of the most common causes for enucleation and carries a significant morbidity rate in affected individuals. Hence, studies on its pathophysiological and growth regulatory mechanisms are urgently needed to identify more effective novel therapeutics. METHODS: Using the Y79 retinoblastoma cell line, we investigated the electrophysiological and functional activities of the T-type voltage-gated calcium channel Cav3.1, that is constitutively expressed in these cells. We also analyzed the Akt and MAPK signaling pathways downstream of the epidermal growth factor receptor (EGFR) to understand the mechanism responsible for the inhibition of Cav3.1. RESULTS: We demonstrate that the EGFR inhibitor Afatinib significantly reduced cell viability and Cav3.1 mRNA expression and electrophysiological activity. At low concentrations (1 µM), Afatinib reduced the amplitude of Cav3.1 current density, whereas at a high concentration (10 µM), it completely abolished the voltage-gated calcium current. Our results show that inhibition of the MAPK pathway by a specific inhibitor VX-11e affected the Cav3.1 current in a dose-dependent manner. VX-11e (50 nM-1 µM) treatment reduced Cav3.1 current densities in Y79 cells, with complete abolishment of Cav3.1 current at higher concentrations (5 µM). We also demonstrate that the specific inhibition of the Akt kinase (using MK-2206) had no effect on the Cav3.1 currents. CONCLUSION: Our study provides a functional relationship between the MAPK pathway and EGFR signaling and indicates that the MAPK signaling pathway mediates the control of Cav3.1 by EGFR in retinoblastoma.

Curcumin blocks Kv11.1 (erg) potassium current and slows proliferation in the infant acute monocytic leukemia cell line THP-1.

Acute Myeloid Leukemia (AML) accounts for approximately one fifth of all childhood leukemia yet is responsible for a significant proportion of morbidity and mortality in this population. For this reason, research to identify novel targets for the development of effective AML therapeutics has intensified in the recent past. The THP-1 cell line, which was originally established from an infant diagnosed with AML, provides an experimental model for functional, pre-clinical therapeutics and target identification studies of AML. Here we show the expression of the voltage gated potassium channel Kv11.1 in THP-1 cells as opposed to normal hematopoietic stem cells. In addition, curcumin, a natural polyphenol derived from the plant Curcuma longa, effectively blocked Kv11.1 activity and also inhibited the proliferation of these cells. Curcumin was rapidly internalized by THP-1 cells and possibly exerts potential growth inhibitory activity by interacting with intracellular epitopes of the ion channel. Inhibition of ionic currents carried by Kv11.1 resulted in depolarization of cell membrane potential. We propose that the inhibition of Kv11.1 activity by curcumin may lead to interference with leukemic cell physiology and consequently the suppression of survival and proliferation of AML cells.

Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition.

BACKGROUND: Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT) constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. RESULTS: AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. CONCLUSIONS: In addition to previously described involvement of insulin like growth factor (IGF) family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

Cytotoxicity, drug combinability, and biological correlates of ABT-737 against acute lymphoblastic leukemia cells with MLL rearrangement.

BACKGROUND: ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity. PROCEDURE: Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis. RESULTS: Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC(50) values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways. CONCLUSIONS: We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials.

Analysis of multiple growth regulatory proteins using dissociable staining antibody arrays on solid tumor biopsy specimens.

Growth of tumor cells is often a function of deregulated growth factor receptors and their corresponding intracellular signalling molecules. The dissociable antibody staining arrays have the versatility to rapidly identify the expression, activation, and localization of such molecules and pathways in biopsy specimens. This report describes a protocol to quantify the activity of a panel of signalling molecules in Wilms tumor biopsy specimens and surrounding nonmalignant renal cells. We propose that this technique can be used to rapidly identify multiple markers and may aid in the study of aberrant growth regulatory mechanisms and potential targets for therapeutics from pathologic specimens.

Cytotoxicity and Target Modulation in Pediatric Solid Tumors by the Proteasome Inhibitor Carfilzomib.

BACKGROUND: Most children with recurrent metastatic solid tumors have high mortality rates. Recent studies have shown that proteasome inhibition leads to effective tumor killing in cells that have acquired treatment resistance and metastatic properties. OBJECTIVE: The purpose of this study was to test the potential of Carfilzomib (CFZ), a proteasome inhibitor, in refractory pediatric solid tumors which is currently unknown. METHODS: A panel of pediatric solid tumor cell lines, including neuroblastoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma and atypical teratoid rhabdoid tumor (ATRT), was used to evaluate the cytotoxic and proteasomal inhibitory effects of CFZ. A drug scheduling experiment was performed to determine the optimal dose and time to obtain effective cell killing. Combination studies of CFZ with chemotherapeutic drugs of different classes were performed to determine the extent of synergy. RESULTS: CFZ showed effective cytotoxicity against all cell lines tested (mean IC50 = 7nM, range = 1-20nM) and activity in a fluorophore-tagged cell-based proteasome assay. Drug scheduling experiments showed that the minimum exposure of 4-8 hours/day is needed for effective cumulative killing. CFZ, when combined with chemotherapeutic drugs of different classes, synergistically enhanced the extent of cell death. CONCLUSION: CFZ showed cytotoxic activity against all the solid pediatric cancer cell lines tested. This study provides initial in vitro data on the potential of CFZ to treat pediatric solid tumors and supports further investigations into the components of drug scheduling, biological correlates and drug combinations for future early phase clinical trials in children.

Human SNF5 arming of double-deleted vaccinia virus shows oncolytic and cytostatic activity against central nervous system atypical teratoid/rhabdoid tumor cells.

Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.

Childhood acute lymphoblastic leukemia (ALL) presenting with severe osteolysis: a model to study leukemia-bone interactions and potential targeted therapeutics.

Interference with the molecular mechanisms that generate tumor supportive niches in the bone microenvironment is a rational approach to inhibit the growth of hematological malignancies. However, the advancement of knowledge in this area has been slowed down by the lack of in vitro models to facilitate the screening of potential candidate agents. The rare cases of acute lymphoblastic leukemia (ALL) in children presenting with extensive bone involvement may represent an exaggerated form of some aspects of the normal tumor-bone interactions. Thus, these cases can provide insight into processes that are otherwise challenging to uncover. The authors describe the case of a 6-year-old child who presented with severe osteopenia that resolved at the time of leukemic remission. Compared to control sera, serum taken at disease presentation contained increased levels of a group of osteolytic cytokines and was effective in activating preosteoclast cells in culture. Based on these findings, the authors describe an experimental model to identify agents that would interfere with leukemia mediated osteolytic process.

The dawn of targeted therapies for triple negative breast cancer (TNBC): a snapshot of investigational drugs in phase I and II trials.

INTRODUCTION: Triple negative breast cancer (TNBC) was once thought to be an insurmountable disease marked by a lack of targeted treatments. However, we are now witnessing the dawn of targeted therapies for TNBC in which progress has stemmed from an improved understanding of the components that make TNBC unique. The identification of biomarkers, such as BRCA1/2, PIK3CA and RSK2, have advanced the field remarkably and there is considerable interest in finding novel therapeutics for TNBC that offer durable clinical benefit with fewer adverse events. AREAS COVERED: We discuss phase I/II trials of new and emerging targeted therapies for TNBC, according to ClinicalTrials.gov up to June 2020. Although the emphasis is on ongoing and completed early phase trials, we also highlight pivotal studies that have led to the approval of new targeted classes of drugs for TNBC, with a focus on outcomes and common adverse events of each class of therapy. EXPERT OPINION: The way forward for TNBC treatment is through precision medicine. The use of novel agents matched with biomarkers to identify patients with the best chance of sustainable response offers new hope. We now have great potential for improving the outcomes for patients with TNBC.

Effects of Hsp90 inhibition in neuroblastoma: analysis of drug sensitivity, target modulation and the influence of bone marrow microenvironment.

Heat shock protein 90 (Hsp90) safeguards the structural integrity and function of many of the key growth regulatory proteins found in malignant cells. Consequently, among the new generation targeted therapeutics, heat shock protein inhibitors have the unique property of being able to target an expansive array of divergent molecular mechanisms involved in cancer growth and metastasis. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) is one such agent that has been shown to bind to Hsp90 and thus reduce the stability and activity of many key growth regulatory molecules and pathways. A number of recent clinical trials have investigated the maximum tolerated dose, toxicity and pharmacokinetic profiles of 17-AAG in pediatric patients with recurrent tumors. In this study, we describe the effects of 17-AAG against a panel of neuroblastoma (NB) cell lines with respect to cytotoxicity, target modulation and inhibition of vascular endothelial growth factor (VEGF) expression. 17-AAG was found to inhibit the growth of all NB cell lines tested, though effective inhibitory concentrations varied among cell lines. 17-AAG also suppressed the expression of VEGF. The cytotoxic effect of 17-AAG on tumor cells was diminished when co-cultured with bone marrow stromal cells suggesting a potential role for the microenvironment in tumor drug interactions. Findings from target modulation analysis as well as drug combination assays provide a frame-work to formulate effective protocols for the treatment of NB.

Targeting the Proteasome in Refractory Pediatric Leukemia Cells: Characterization of Effective Cytotoxicity of Carfilzomib.

BACKGROUND: Leukemia accounts for 30% of all childhood cancers and although the survival rate for pediatric leukemia has greatly improved, relapse is a major cause of treatment failure. Therefore, the development and introduction of novel therapeutics to treat relapsed pediatric leukemia is urgently needed. The proteasome inhibitor bortezomib has been shown to be effective against adult hematological malignancies such as multiple myeloma and lymphoma, but is frequently associated with the development of resistance. Carfilzomib is a next-generation proteasome inhibitor that has shown promising results against refractory adult hematological malignancies. OBJECTIVE: Carfilzomib has been extensively studied in adult hematological malignancies, providing the rationale for evaluating proof-of-concept activity of carfilzomib in pediatric leukemia. METHODS: The effects of carfilzomib on pediatric leukemia cell lines and primary pediatric leukemia patient samples were investigated in vitro using the alamar blue cytotoxicity assay, western blotting, and a proteasome activity assay. Synergy with commonly used anticancer drugs was determined by calculation of combination indices. RESULTS: In vitro preclinical data show pharmacologically relevant concentrations of carfilzomib are cytotoxic to pediatric leukemia cell lines and primary pediatric leukemia cells. Target modulation studies validate the effective inhibition of the proteasome and induction of apoptosis. We also identify agents that have effective synergy with carfilzomib in these cells. CONCLUSIONS: Our data provide pre-clinical information that can be incorporated into future early-phase clinical trials for the assessment of carfilzomib as a treatment for children with refractory hematological malignancies.

In Vitro Sensitivity Profiling of Neuroblastoma Cells Against A Comprehensive Small Molecule Kinase Inhibitor Library to Identify Agents for Future Therapeutic Studies.

BACKGROUND: Neuroblastoma (NB) constitutes about 8% of all childhood tumors, yet accounts for more than 15% of deaths, with an unacceptable overall survival rate. These rates are despite the current multimodal therapeutic approaches involving surgery, radiation, chemotherapy and myeloablation with hematopoietic stem cell rescue. Hence, efforts have intensified to identify new targets and novel therapeutic approaches to improve cure rates in these children. Numerous new agents for adult malignancies are developed and evaluated for cancer each year, providing an invaluable resource, with the added advantage of available pharmacologic and toxicity data for consideration. METHODS: To identify potential therapeutic targets, we screened a small molecule library of 151 small kinase inhibitors against NB cell lines. Based on our initial screening data, we further examined the potential of Bcr-Abl targeting small molecule inhibitors to affect the growth and survival of NB cells. RESULTS: There is diverse activity among the currently available Bcr-Abl inhibitors, possibly reflecting the molecular heterogeneity and off-target activity in each combination. In depth analyses of ponatinib, an oral multi-target kinase inhibitor and effective agent in the treatment of refractory Philadelphia chromosome (Ph) positive leukemia, show growth inhibition at sub-micromolar concentrations. In addition, we also identified the potential of this agent to interfere with insulin-like growth factor-1 receptor (IGF-1R) signaling pathways and Src activity, inhibit cell migration and induce apoptosis. CONCLUSION: Our findings provide initial data on ponatinib's potential to target key growth regulatory pathways and provide the rationale for further studies and evaluation in future early phase clinical trials for the treatment of refractory NB.

Druggable targets in pediatric neurocutaneous melanocytosis: Molecular and drug sensitivity studies in xenograft and ex vivo tumor cell culture to identify agents for therapy.

BACKGROUND: Neurocutaneous melanocytosis (NCM) is a rare congenital disorder that presents with pigmented cell lesions of the brain or leptomeninges in children with large or multiple congenital melanocytic nevi. Although the exact pathological processes involved are currently unclear, NCM appears to arise from an abnormal development of melanoblasts or melanocyte precursors. Currently, it has an extremely poor prognosis due to rapid disease progression and lack of effective treatment modalities. METHODS: In this study, we report on an experimental approach to examining NCM cells by establishing subcutaneous tumors in nude mice, which can be further expanded for conducting molecular and drug sensitivity experiments. RESULTS: Analysis of the NRAS gene-coding sequences of an established NCM cell line (YP-MEL) and NCM patient cells revealed heterogeneity in NRAS Q61K that activated mutation and possibly consequential differential sensitivity to MEK inhibition. Gene expression studies were performed to compare the molecular profiles of NCM cells with normal skin fibroblasts. In vitro cytotoxicity screens of libraries of targeted small-molecule inhibitors revealed prospective agents for further evaluation. CONCLUSIONS: Our studies provide an experimental platform for the generation of NCM cells for preclinical studies and the production of molecular and in vitro data with which to identify druggable targets for the treatment.