Kristen M. Smith

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.

Neuroblastoma Cells Isolated from Bone Marrow Metastases Contain a Naturally Enriched Tumor-Initiating Cell

Neuroblastoma is a heterogeneous pediatric tumor thought to arise from the embryonic neural crest. Identification of the cell responsible for propagating neuroblastomas is essential to understanding this often recurrent, rapidly progressing disease. We have isolated and characterized putative tumor-initiating cells from 16 tumors and bone marrow metastases from patients in all neuroblastoma risk groups. Dissociated cells from tumors or bone marrow grew as spheres in conditions used to culture neural crest stem cells, were capable of self-renewal, and exhibited chromosomal aberrations typical of neuroblastoma. Primary spheres from all tumor risk groups differentiated under neurogenic conditions to form neurons. Tumor spheres from low-risk tumors frequently formed large neuronal networks, whereas those from high-risk tumors rarely did. As few as 10 passaged tumor sphere cells from aggressive neuroblastoma injected orthotopically into severe combined immunodeficient/Beige mice formed large neuroblastoma tumors that metastasized to liver, spleen, contralateral adrenal and kidney, and lung. Furthermore, highly tumorigenic tumor spheres were isolated from the bone marrow of patients in clinical remission, suggesting that this population of cells may predict clinical behavior and serve as a biomarker for minimal residual disease in high-risk patients. Our data indicate that high-risk neuroblastoma contains a cell with cancer stem cell properties that is enriched in tumor-initiating capacity. These cells may serve as a model system to identify the molecular determinants of neuroblastoma and to develop new therapeutic strategies for this tumor.

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Selective targeting of neuroblastoma tumour‐initiating cells by compounds identified in stem cell‐based small molecule screens

Neuroblastoma (NB) is the most deadly extra‐cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour‐initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient‐derived cancer stem cell‐like tumour‐initiating cells (TICs) while sparing normal paediatric stem cells (skin‐derived precursors, SKPs) and characterize two therapeutic candidates. DECA‐14 and rapamycin were identified as NB TIC‐selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self‐renewal or tumour‐initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient‐specific and potentially less toxic therapies.

System-Level Analysis of Neuroblastoma Tumor–Initiating Cells Implicates AURKB as a Novel Drug Target for Neuroblastoma

Purpose: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. Experimental Design: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. Results: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. Conclusion: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets. Clin Cancer Res; 16(18); 4572–82. ©2010 AACR.

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Background Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated. Methodology and Principal Findings We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34+ cells from NOTCH1Mutated T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1Wild-type CD34+ cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1Mutated CD34+ fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1Mutated T-ALL LIC-engrafted mice and resulted in depletion of CD34+CD2+CD7+ cells that harbor serial transplantation capacity. Conclusions These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.

A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions

Prostate cancer metastasizes to bone in the majority of patients with advanced disease leading to painfully debilitating fractures, spinal compression and rapid decline. In addition, prostate cancer bone metastases often become resistant to standard therapies including androgen deprivation, radiation and chemotherapy. There are currently few models to elucidate mechanisms of interaction between the bone microenvironment and prostate cancer. It is, thus, essential to develop new patient-derived, orthotopic models. Here we report the development and characterization of PCSD1 (Prostate Cancer San Diego 1), a novel patient-derived intra-femoral xenograft model of prostate bone metastatic cancer that recapitulates mixed osteolytic and osteoblastic lesions.MethodsA femoral bone metastasis of prostate cancer was removed during hemiarthroplasty and transplanted into Rag2-/-;γc-/- mice either intra-femorally or sub-cutaneously. Xenograft tumors that developed were analyzed for prostate cancer biomarker expression using RT-PCR and immunohistochemistry. Osteoblastic, osteolytic and mixed lesion formation was measured using micro-computed tomography (microCT).ResultsPCSD1 cells isolated directly from the patient formed tumors in all mice that were transplanted intra-femorally or sub-cutaneously into Rag2-/-;γc-/- mice. Xenograft tumors expressed human prostate specific antigen (PSA) in RT-PCR and immunohistochemical analyses. PCSD1 tumors also expressed AR, NKX3.1, Keratins 8 and 18, and AMACR. Histologic and microCT analyses revealed that intra-femoral PCSD1 xenograft tumors formed mixed osteolytic and osteoblastic lesions. PCSD1 tumors have been serially passaged in mice as xenografts intra-femorally or sub-cutaneously as well as grown in culture.ConclusionsPCSD1 xenografts tumors were characterized as advanced, luminal epithelial prostate cancer from a bone metastasis using RT-PCR and immunohistochemical biomarker analyses. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely resembled the bone lesions in the patient. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study metastatic disease in the bone and to develop novel therapies for inhibiting prostate cancer growth in the bone-niche.

Identification of Drugs that Regulate Dermal Stem Cells and Enhance Skin Repair

Summary Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs), a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Antitumor Activity of Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor, in ETV6-NTRK3–Positive Acute Myeloid Leukemia

Activation of tropomyosin receptor kinase (TRK) family tyrosine kinases by chromosomal rearrangement has been shown to drive a wide range of solid tumors and hematologic malignancies. TRK fusions are actionable targets as evidenced by recent clinical trial results in solid tumors. Entrectinib (RXDX-101) is an investigational, orally available, CNS-active, highly potent, and selective kinase inhibitor against TRKA/B/C, ROS1, and ALK kinase activities. Here, we demonstrate that TRK kinase inhibition by entrectinib selectively targets preclinical models of TRK fusion–driven hematologic malignancies. In acute myelogenous leukemia (AML) cell lines with endogenous expression of the ETV6–NTRK3 fusion gene, entrectinib treatment blocked cell proliferation and induced apoptotic cell death in vitro with subnanomolar IC50 values. Phosphorylation of the ETV6–TRKC fusion protein and its downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. In animal models, entrectinib treatment at clinically relevant doses resulted in tumor regression that was accompanied by elimination of residual cancer cells from the bone marrow. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with TRK fusion–driven AML and other hematologic malignancies. Mol Cancer Ther; 17(2); 455–63. ©2017 AACR.

A Phosphoproteomics Approach to Identify Candidate Kinase Inhibitor Pathway Targets in Lymphoma-Like Primary Cell Lines

Mass spectrometry-based technologies are increasingly utilized in drug discovery. Phosphoproteomics in particular has allowed for the efficient surveying of phosphotyrosine signaling pathways involved in various diseases states, most prominently in cancer. We describe a phosphotyrosine-based proteomics screening approach to identify signaling pathways and tyrosine kinase inhibitor targets in highly tumorigenic human lymphoma-like primary cells. We identified several receptor tyrosine kinase pathways and validated SRC family kinases (SFKs) as potential drug targets for targeted selection of small molecule inhibitors. BMS-354825 (dasatinib) and SKI-606 (bosutinib), second and third generation clinical SFK/ABL inhibitors, were found to be potent cytotoxic agents against tumorigenic cells with low toxicity to normal pediatric stem cells. Both SFK inhibitors reduced ERK1/2 and AKT phosphorylation and induced apoptosis. This study supports the adaptation of high-end mass spectrometry techniques for the efficient identification of candidate tyrosine kinases as novel therapeutic targets in primary cancer cell lines.