Jeanne Kowalski

Concise review: Emerging concepts in clinical targeting of cancer stem cells

Cancer stem cells (CSCs) are functionally defined by their ability to self‐renew and recapitulate tumors in the ectopic setting. They have been identified in a growing number of human malignancies and their association with poor clinical outcomes has suggested that they are the major factors in dictating clinical outcomes. Moreover, recent studies have demonstrated that CSCs may display other functional attributes, such as drug resistance and invasion and migration, that implicate a broad role in clinical oncology spanning initial tumor formation, relapse following treatment, and disease progression. Although our knowledge regarding the basic biology of CSCs continues to improve, proof that they are clinically relevant is still lacking, and translation of the CSC hypothesis from the laboratory to the clinic is of paramount importance. We will review current evidence supporting the role of CSCs in clinical oncology and discuss potential barriers and strategies in designing trials examining CSC‐targeting agents. STEM CELLS 2011;29:883–887

K562/GM-CSF Immunotherapy Reduces Tumor Burden in Chronic Myeloid Leukemia Patients with Residual Disease on Imatinib Mesylate

Purpose: Chronic myeloid leukemia (CML) can be responsive to T-cell–mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a GM-CSF producing vaccine derived from a CML cell line that expresses several CML-associated antigens. A pilot study was developed to determine if K562/GM-CSF immunotherapy could improve clinical responses to imatinib mesylate (IM) in patients with chronic myeloid leukemia. Experimental Design: Patients with chronic phase CML who achieved at least a major cytogeneic response but remained with persistent, measurable disease despite one or more years on imatinib mesylate were eligible. Each was given a series of four vaccines administered in three-week intervals, with or without topical imiquimod, while remaining on a stable dose of imatinib mesylate. CML disease burden was measured serially before and after vaccination. Results: Nineteen patients were vaccinated, with a median duration of previous imatinib mesylate therapy of 37 (13–53) months. Mean PCR measurements of BCR-ABL for the group declined significantly following the vaccines (P = 0.03). Thirteen patients had a progressive decline in disease burden, 8 of whom had increasing disease burden before vaccination. Twelve patients achieved their lowest tumor burden measurements to date following vaccine, including seven subjects who became PCR-undetectable. Conclusions: K562/GM-CSF vaccine appears to improve molecular responses in patients on imatinib mesylate, including achieving complete molecular remissions, despite long durations of previous imatinib mesylate therapy. Clin Cancer Res; 16(1); 338–47

CCAAT/enhancer binding protein α (C/EBPα) and C/EBPα myeloid oncoproteins induce Bcl-2 via interaction of their basic regions with nuclear factor-κB p50

The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein α (C/EBPα) or its mutant variants contributes to transformation. C/EBPαp30, lacking a NH2-terminal transactivation domain, or C/EBPαLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPα induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPα-Eμ transgenic mice. C/EBPα protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPαLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPαp30 also activated the promoter. C/EBPα and the C/EBPα oncoproteins cooperated with nuclear factor-κB (NF-κB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPα preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPα basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPα or its mutant variants tether to a subset of NF-κB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPαLZ oncoproteins. Targeting interaction between C/EBPα basic region and NF-κB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.

Pronecrotic mixed lineage kinase domain-like protein expression is a prognostic biomarker in patients with early-stage resected pancreatic adenocarcinoma

Mixed lineage kinase domain‐like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC).

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma

Background:It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT).Methods:DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs.Results:We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-β and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-β and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs.Conclusions:Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring.

Non-parametric, hypothesis-based analysis of microarrays for comparison of several phenotypes

MOTIVATION We present a statistical framework for the analysis of high-dimensional microarray data, where the goal is to compare intensities among several groups based on as few as a single sample from each group. In this setting, it is of interest to compare gene expression among several phenotypes to define candidate genes that simultaneously characterize several criteria, simultaneously, among the comparison groups. We motivate the approach by a comparative microarray experiment in which clones of a cell were singly exposed to several distinct but related conditions. The experiment was conducted to elucidate genes involved in pathways leading to T cell clonal anergy. RESULTS By integrating inference principles within a bioinformatics setting, we introduce a two-stage approach to select candidate genes that characterize several criteria. The method is unified in its non-parametric approach to inference and description. For inference, we construct a testable hypothesis based on the criteria of interest in a high-dimensional space, while preserving the dependence among genes. Upon rejecting the null, we estimate the cardinality of a set of individual candidate genes (or gene pairs) that depict the events of interest. With this estimate, we then select individual genes (or gene pairs) based upon a two-dimensional ranking that examines relations within and between genes, among comparison groups, using singular value decomposition in combination with inner product concepts.

Class I HDACs are mediators of smoke carcinogen-induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer.

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention. Cancer Prev Res; 7(3); 351–61. ©2014 AACR.

Cyclooxygenase inhibitors block uterine tumorigenesis in HMGA1a transgenic mice and human xenografts

Uterine cancer is a common cause for cancer death in women and there is no effective therapy for metastatic disease. Thus, research is urgently needed to identify new therapeutic agents. We showed previously that all female HMGA1a transgenic mice develop malignant uterine tumors, indicating that HMGA1a causes uterine cancer in vivo. We also demonstrated that HMGA1a up-regulates cyclooxygenase-2 (COX-2) during tumorigenesis in this model. Similarly, we found that HMGA1a and COX-2 are overexpressed in human leiomyosarcomas, a highly malignant uterine cancer. Although epidemiologic studies indicate that individuals who take COX inhibitors have a lower incidence of some tumors, these inhibitors have not been evaluated in uterine cancer. Here, we show that HMGA1a mice on sulindac (a COX-1/COX-2 inhibitor) have significantly smaller uterine tumors than controls. To determine if COX inhibitors are active in human uterine cancers that overexpress HMGA1a, we treated cultured cells with sulindac sulfide or celecoxib (a specific COX-2 inhibitor). Both drugs block anchorage-independent growth in high-grade human uterine cancer cells that overexpress HMGA1a (MES-SA cells). In contrast, neither inhibitor blocked transformation in cells that do not overexpress HMGA1a. Moreover, xenograft tumors from MES-SA cells were significantly inhibited in mice on sulindac. More strikingly, no tumors formed in mice on celecoxib. These preclinical studies suggest that COX inhibitors could play a role in preventing tumor onset or progression in uterine cancers with dysregulation of the HMGA1a-COX-2 pathway. Importantly, these drugs have lower toxicity than chemotherapeutic agents used to treat advanced-stage uterine cancers. [Mol Cancer Ther 2008;7(7):2090–5]

Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer.

Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single‐agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA‐PKcs protein modulation. Gene expression profiling identified differential expression of a 5‐gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell‐specific manner. This potentiation correlates with platinum sensitivity, DNA‐PKcs expression and a 5‐gene expression profile.

High Dose Cyclophosphamide without Stem Cell Rescue in 207 Patients with Aplastic anemia and other Autoimmune Diseases

High-dose cyclophosphamide has long been used as an anticancer agent, a conditioning regimen for hematopoietic stem cell transplantation, and a potent immunosuppressive agent in autoimmune diseases including aplastic anemia. High-dose cyclophosphamide is highly toxic to lymphocytes but spares hematopoietic stem cells because of their abundant levels of aldehyde dehydrogenase, the major mechanism of cyclophosphamide inactivation. High-dose cyclophosphamide therapy induces durable remissions in most patients with acquired aplastic anemia. Moreover, high-dose cyclophosphamide without hematopoietic stem cell rescue has shown activity in a variety of other severe autoimmune diseases.Here we review the history of cyclophosphamide as it applies to aplastic anemia and other autoimmune diseases. We include historical data from early patients treated for aplastic anemia as well as data from 140 patients from an observational retrospective study in a single tertiary care hospital. This latter component was designed to assess the safety and efficacy of high-dose cyclophosphamide therapy without stem cell rescue in patients with refractory autoimmune diseases. We analyzed the 140 patients with severe, progressive autoimmune diseases treated. All patients discussed here received cyclophosphamide, 50 mg/kg per day for 4 consecutive days. Response, relapse, and overall survival were measured. Response was defined as a decrease in disease activity in conjunction with a decrease or elimination of immune-modulating drugs. Relapse was defined as worsening disease activity and/or a requirement for an increase in dose of, or administration of new, immunosuppressive medications.Hematologic recovery occurred in all patients. The overall response rate was 94%, and 44% of those patients remained progression free with a median follow-up of 36 months (range, 1-120 mo) for the 140 patients analyzed together. The overall actuarial and event-free survival across all diseases at 60 months was 90.7% and 20.6%, respectively. High-dose cyclophosphamide without stem cell rescue is well tolerated and induces a high rate of remission in severe autoimmune diseases.Abbreviations: ATG = antithymocyte globulin, CIBMTR = Center for International Blood and Marrow Transplant Research, CsA = cyclosporine A, EBMT = European Group for Blood and Marrow Transplantation, EDSS = Expanded Disability Status Scale, EULAR = European League Against Rheumatism, HLA = human leukocyte antigen, HSCT = hematopoietic stem cell transplantation, MDS = myelodysplastic syndrome, PNH = paroxysmal nocturnal hemoglobinuria, SAA = severe aplastic anemia, SLE = systemic lupus erythematosus.