Abstract Introduction: Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma. An aggressive subtype, fusion-positive RMS (FP-RMS), which is driven by PAX3/7::FOXO1 translocations, has a dismal 5-year overall survival rate of 13% for patients with metastatic disease. We hypothesized that targeting the oncogenic driver PAX3::FOXO1 with small molecules would be an effective treatment. To test this, we developed cell lines with endogenous PAX3::FOXO1 tagged with HiBiT epitope and performed a drug screen to identify drugs that downregulate PAX3::FOXO1 protein. Study Design: Using CRISPR-Cas9, we endogenously tagged PAX3::FOXO1 with HiBiT in two RMS cell lines (RH4 and SCMC), enabling the monitoring of PAX3::FOXO1 protein levels. We performed a drug screen using the Mechanism Interrogation Plate (MIPE) library of 2,480 compounds, of which 53% are FDA-approved or in clinical trials. NanoGlo Luciferase assays monitored levels of HiBiT-tagged PAX3::FOXO1, while CellTiterGlo measured cell viability at 24 hours. We selected hits that showed a difference in area under the curve (AUC) between the two readouts of ≥ 90 for drugs that preferentially reduce the fusion protein level over general cytotoxicity. We investigated whether inhibitors led to nuclear accumulation and reduced total protein levels using Western blot and immunofluorescent imaging. Candidates were validated in the parental cells and in vivo studies. Results and Conclusions: The screen identified 183 hits, including Eltanexor, an XPO1 inhibitor. XPO1 exports over 200 proteins from the nucleus by recognizing their nuclear export sequences (NESs). Since the fusion gene retains the NES of FOXO1, a known XPO1 target, we tested whether PAX3::FOXO1 is a substrate. We observed that Eltanexor enhanced PAX3::FOXO1 nuclear accumulation at 6 hours in RH4, and at 2 hours in SCMC, followed by protein downregulation at 24 hours by Western blotting. Furthermore, Eltanexor induced p53 nuclear accumulation, detectable at 6 hours in SCMC, suggesting that early accumulation of PAX3::FOXO1 may drive cytotoxicity. At 24 hours, RNA-seq in Eltanexor-treated cell lines demonstrated downregulation of PAX3::FOXO1 and MYCN signatures, components of the core regulatory network in FP-RMS. Preliminary in vivo studies also showed Eltanexor induces delays in tumor progression in an RMS xenograft model. Furthermore, Eltanexor in combination with Mivebresib, a validated BRD4 inhibitor, demonstrated significant synergy against FP-RMS cells. We will perform site-directed mutagenesis studies to disrupt PAX3::FOXO1’s NES and validate combination with Mivebresib in vivo. In conclusion, we identified Eltanexor, an XPO1 inhibitor, as a novel therapeutic agent that suppressed PAX3::FOXO1 activity and levels, induced nuclear accumulation and led to cytotoxicity in incurable FP-RMS. Citation Format: Soumili Dey, Yong Y. Kim, Katrina Jia, Mehal Churiwal, Michele Ceribelli, Teresa S. Hawley, Raj Chari, David Milewski, Young K. Song, Xinyu Wen, Hsien-Chao Chou, Vineela Gangalapudi, Jun S. Wei, Craig Thomas, Robert G. Hawley, Javed Khan. Small-molecule screening of HiBiT-tagged PAX3::FOXO1 rhabdomyosarcoma cell lines identifies eltanexor as a potent therapeutic agent against fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6405.

Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancer. PAX3::FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the C-terminal end of the endogenous P3F using CRISPR. Western was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen was performed using the Mechanism Interrogation PlatE drug library with known mechanisms of action. Cell viability was measured using CellTiter-Glo. Mouse xenograft models were used to investigate in vivo efficacy. We validated the HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody verified that P3F-HiBiT binds to the same sites as P3F. A screen for compounds that downregulate P3F in both RH4 and SCMC identified 182 drugs. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes, including HDAC inhibitors, BRD4 inhibitors, and CDK inhibitors. Focusing on CDK inhibitors, we found that FP-RMS was most sensitive to CDK7, CDK9 and multi-CDK inhibitors. TG02, a multi-CDK inhibitor with highest inhibition of CDK9 and currently in human trials, downregulated P3F protein. GSEA showed marked suppression of P3F targets after TG02 treatment. Western validated the inhibition of CDK9 with decreased RNA Pol2 Ser2 phosphorylation (Pol2S2). ChIP-seq for RNA Pol2 showed a decrease in transcription pause-release, indicating inhibition of transcription by TG02. Moreover, analysis of genes ranked by decreased Pol2S2 in the gene body showed significant enrichment for P3F targets (p<0.001). TG02 significantly delayed tumor progression without weight loss in a mouse xenograft model of FP-RMS. Also, we found that Vincristine (VCR) and Irinotecan (IRN) are synergistic with TG02 in vitro. Combinations of TG02 with VCR or IRN showed a significant delay in tumor progression compared to TG02 alone in mouse xenograft models. By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels. One of the top hits, TG02, showed in vivo efficacy, indicating that FP-RMS is susceptible to multi-CDK inhibition. Decreased occupancy of Pol2S2 in the gene body of P3F targets indicates that the mechanism of TG02 is primarily through transcriptional inhibition of P3F and its targets. This indicates that TG02 may be effective in transcriptionally addicted cancers such as FP-RMS. We also found synergy between TG02 with VCR and IRN showing promise for clinical translation in FP-RMS. Yong Yean Kim, Katrina Jia, Mehal Churiwal, Soumili Dey, Teresa S. Hawley, Silvia Pomella, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Craig J. Thomas, Michele Ceribelli, Jun S. Wei, Robert G. Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3::FOXO1 reveals that CDK inhibitors downregulate the fusion oncogene, and demonstrate synergy effects when combined with vincristine and irinotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7037.

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens to identify suppressors of P3F. Methods: HiBiT tag, an 11 amino acid peptide of the small fragment of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR-Cas9 in FP-RMS cell lines RH4 and SCMC. Western analysis was used for HiBiT tag validation and confirmation of P3F suppression. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT) respectively. High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, which included 2,480 drugs with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. We identified drugs that suppressed P3F by Nano-Glo without acute cytotoxicity by CellTiter-Glo at an early 24-hour timepoint. Mouse xenograft model of FP-RMS was used to investigate in vivo efficacy of top hits. Results: We validated HiBiT tagging of P3F and not the wild-type FOXO1 by Western analysis. We showed that the HiBiT tag did not change the function of P3F by transducing human fibroblasts with P3F-HiBiT versus unmodified P3F. Gene Set Enrichment Analysis (GSEA) of RNA-seq showed that P3F-HiBiT activated the same downstream target genes as unmodified P3F. ChIP-seq using HiBiT antibody in HiBiT-tagged FP-RMS cell lines RH4 and SCMC matched the genomic locations from ChIP-seq with P3F antibody in parental RH4 and SCMC. Using a cutoff of Area Under the Curve (AUC) of CellTiter-Glo - AUC of Nano-Glo > 90, in both RH4 and SCMC, identified 182 compounds. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F protein level including HDAC inhibitors (3), mTOR inhibitors (4), CDK inhibitors (8), and BRD4 inhibitors (3). One top hit was the CDK inhibitor TG02 (Zotiraciclib), currently in human trials. TG02 suppressed P3F protein levels by Nano-Glo and Western analysis. We confirmed induction of apoptosis by PARP cleavage in a panel of FP-RMS cell lines. GSEA analysis of RNA-seq after treatment with TG02 showed marked suppression of P3F target gene sets. TG02 also significantly delayed tumor progression of established tumors in a mouse xenograft model of FP-RMS without weight loss. Conclusion and Future Directions:By HiBiT tagging the fusion oncogene P3F, we identified 182 compounds that suppress P3F levels of which TG02 was a top hit that also showed in vivo efficacy. Drug combination studies are currently underway to identify synergistic suppressors of P3F protein levels that can be translated into clinical trials. Citation Format: Yong Yean Kim, Robert G. Hawley, Mehal Churiwal, Teresa S. Hawley, Christine N. Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Ying Pang, Jing Wu, Craig J. Thomas, Jun S. Wei, Michele Ceribelli, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3538.

The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer (SpTrf ; formerly Sp185/333) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

Sea urchin coelomocytes can be collected in large numbers from adult sea urchins of the species, Strongylocentrotus purpuratus, which typically has 12-40mL of coelomic fluid. Coelomocytes are used for analysis of immune reactions and immune gene expression in addition to basic functions of cells, in particular for understanding structure and modifications of the cytoskeleton in phagocytes. The methods described here include coelomocyte isolation, blocking the clotting reaction, establishing and maintaining primary cultures, separation of different types of coelomocytes into fractions, processing live coelomocytes for light microscopy, fixation and staining for light and electron microscopy, analysis of coelomocyte populations by flow cytometry, and sorting single cells for more detailed follow-up analyses including transcriptomics or genomic characteristics. These methods are provided to make working with coelomocytes accessible to researchers who are unfamiliar with these cells and perhaps to aid others who have worked extensively with invertebrate cells.

Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease. However, MM cells still develop resistance to proteasome inhibitors such as carfilzomib. Toward this end, we have established carfilzomib-resistant derivatives of MM cell lines. We found that resistance to carfilzomib was associated with elevated levels of prosurvival autophagy, and Kruppel-like factor 4 (KLF4) was identified as a contributing factor. Expression levels as well as nuclear localization of KLF4 protein were elevated in MM cells with acquired carfilzomib resistance. Chromatin immunoprecipitations indicated that endogenous KLF4 bound to the promoter regions of the SQSTM1 gene encoding the ubiquitin-binding adaptor protein sequestosome/p62 that links the proteasomal and autophagic protein degradation pathways. Ectopic expression of KLF4 induced upregulation of SQSTM1. On the other hand, inhibitors of autophagy sensitized MM cells to carfilzomib, even in carfilzomib-resistant derivatives having increased expression of the multidrug resistance protein P-glycoprotein. Thus, we report here a novel function for KLF4, one of the Yamanaka reprogramming factors, as being a contributor to autophagy gene expression which moderates preclinical proteasome inhibitor efficacy in MM.

Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b(-) CD8(+) DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b(-) CD8(+) DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103(+) CD11b(-) CD8(+) DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103(+) CD11b(-) CD8(+) DC isolated from infected animals in the generation of an IEL response against a live pathogen.

Analysis of cells via flow cytometry requires calibration of the instrument with the fluorophore used to label the cells of interest. Commercially-available calibration beads are an indispensable tool when preparing flow cytometers for experiments using fluorescent dyes (e.g. FITC); however, due to the different spectral characteristics of fluorescent proteins versus fluorescent dyes, existing fluorescent dye beads are not suitable for instrument calibration if the cells being analyzed express fluorescent proteins. Therefore, we developed calibration beads labeled with either the red fluorescent protein mCherry or the green fluorescent protein AcGFP, which has spectral properties almost identical to EGFP. Beads with a very low size deviation (CV 2.5–3%) were used to create distinct fluorescent bead populations by covalently linking specific amounts of the respective fluorescent proteins. The low size deviation of the beads, in conjunction with a very controlled labeling method, allowed us to create six bead populations with distinct fluorescent intensities for each of the two fluorescent proteins. Here, we show that the fluorescent protein Flow Cytometer Calibration Beads are easy to use and that they perform equally well on a variety of flow cytometer platforms. We also present data showing that the mean fluorescence intensity of the beads and the calculated number of fluorescent proteins on each respective bead population are distinct from each other and in a linear correlation. We also provide supporting data showing the signal stability of the calibration beads under different buffer and fixative conditions, as well as at different flow rates. The data show that these calibration beads are a very useful tool, enabling fast and reliable calibration of flow cytometers prior to analysis of cells expressing the corresponding fluorescent protein.

INTRODUCTION:Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency.METHODS:To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector.RESULTS:Retrovirally transduced TLX1-expressing murine bone marrow progenitor cells had a growth/survival advantage and readily gave rise to immortalized cell lines. Extensive characterization of 15 newly established cell lines failed to reveal a common retroviral integration site. This comprehensive analysis greatly extends our previous study involving a limited number of cell lines, providing additional support for the view that constitutive TLX1 expression is sufficient to initiate the series of events culminating in hematopoietic progenitor cell immortalization. When TLX1-immortalized cells were co-cultured on OP9-DL1 monolayers under conditions permissive for T-cell differentiation, a latent T-lineage potential was revealed. However, the cells were unable to transit the DN2 myeloid-T (DN2mt)-DN2 T-lineage determined (DN2t) commitment step. The differentiation block coincided with failure to upregulate the zinc finger transcription factor gene Bcl11b, the human ortholog of which was shown to be a direct transcriptional target of TLX1 downregulated in the TLX1+ T-ALL cell line ALL-SIL. Other studies have described the ability of TLX1 to promote bypass of mitotic checkpoint arrest, leading to aneuploidy. We likewise found that diploid TLX1-expressing DN2mt cells treated with the mitotic inhibitor paclitaxel bypassed the mitotic checkpoint and displayed chromosomal instability. This was associated with elevated expression of TLX1 transcriptional targets involved in DNA replication and mitosis, including Ccna2 (cyclin A2), Ccnb1 (cyclin B1), Ccnb2 (cyclin B2) and Top2a (topoisomerase IIα). Notably, enforced expression of BCL11B in ALL-SIL T-ALL cells conferred resistance to the topoisomerase IIα poison etoposide.CONCLUSION:Taken together with previous findings, the data reinforce a mechanism of TLX1 oncogenic activity linked to chromosomal instability resulting from dysregulated expression of target genes involved in mitotic processes. We speculate that repression of BCL11B expression may provide part of the explanation for the observation that aneuploid DNA content in TLX1+ leukemic T cells does not necessarily portend an unfavorable prognosis. This TLX1 hematopoietic progenitor cell immortalization/T-cell differentiation assay should help further our understanding of the mechanisms of TLX1-mediated evolution to malignancy and has the potential to be a useful predictor of disease response to novel therapeutic agents in TLX1+ T-ALL.

Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy‐resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P‐glycoprotein, a member of the ABC (ATP‐binding cassette) superfamily of transporters encoded by ABCB1 . We also demonstrate that ABCB1 ‐overexpressing MM cells are resistant to the second‐generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy‐refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell‐surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1 ‐mediated drug resistance should it occur. Am. J. Hematol. 88:265–272, 2013. © 2013 Wiley Periodicals, Inc.

Cold atmospheric plasma (CAP), a technology based on quasi-neutral ionized gas at low temperatures, is currently being evaluated as a new highly selective alternative addition to existing cancer therapies. Here, we present a first attempt to identify the mechanism of CAP action. CAP induced a robust ~2-fold G2/M increase in two different types of cancer cells with different degrees of tumorigenicity. We hypothesize that the increased sensitivity of cancer cells to CAP treatment is caused by differences in the distribution of cancer cells and normal cells within the cell cycle. The expression of γH2A.X (pSer139), an oxidative stress reporter indicating S-phase damage, is enhanced specifically within CAP treated cells in the S phase of the cell cycle. Together with a significant decrease in EdU-incorporation after CAP, these data suggest that tumorigenic cancer cells are more susceptible to CAP treatment.

Fluorescent proteins are now a critical tool in all areas of biomedical research. In this article, we review the techniques required to use fluorescent proteins for flow cytometry, concentrating specifically on the excitation and emission requirements for each protein, and the specific equipment required for optimal use.

Despite considerable progress in the treatment of T cell acute lymphoblastic leukemia (T-ALL), it is still the highest risk malignancy among ALL. The outcome of relapsed patients remains dismal. The pro-survival role of NOTCH1 and NFkB in T-ALL is well documented; also, both factors were reported to be predictive of relapse. The NOTCH1 signaling pathway, commonly activated in T-ALL, was shown to enhance the transcriptional function of NFkB via several mechanisms. Thus, pharmacological inhibition of NOTCH1-NFkB signaling was suggested to be incorporated into existing T-ALL treatment protocols. However, conventional chemotherapy is based on activation of various types of stress, such as DNA damage, mitotic perturbations or endoplasmic reticulum overload. NFkB is frequently activated in response to stress and, depending on yet unknown mechanisms, it either protects cells from the drug action or mediates apoptosis. Here, we report that T-ALL cells respond to NFkB inhibition in opposite ways depending on whether they were treated with a stress-inducing chemotherapeutic agent or not. Moreover, we found that NOTCH1 enhances NFkB apoptotic function in the stressed cells. The data argue for further studies of NFkB status in T-ALL patients on different treatment protocols and the impact of activating NOTCH1 mutations on treatment response. Mol Cancer Res; 9(8); 979–84.

Exposure to ultraviolet (UV) radiation has been implicated in melanoma formation. Glenn Merlino and colleagues now show, in a mouse model, that UVB irradiation induces the recruitment of macrophages that produce interferon-γ to promote melanoma formation. The study suggests that agents that target interferon-γ may have therapeutic potential in patients with melanoma. Ultraviolet (UV) exposure has been implicated in melanoma formation. Here it is shown, in a mouse model, that UVB induces the recruitment of macrophages which produce interferon-γ to promote melanomagenesis. The study suggests that in melanomas, interferon-γ could be targeted therapeutically. Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.