FACT (Facilitates chromatin transcription) complex as a ... · SPT16 SSRP1 b-actin FACT...
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SPT16 SSRP1 b-actin FACT (Facilitates chromatin transcription) complex as a marker and target of aggressive poorly differentiated cancers Henry Garcia 1 , Alfiya Safina 1 , Daria Fleyshman 1 , Catherine Burkhart 4, , Andrei Purmal 5, Jeffrey C Miecznikowski 3 , Angela R. Omilian 2 , Carl Morrison 2 , Katerina V Gurova 1 1 Department of Cell Stress Biology, 2 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, 3 Department of Biostatistics, SUNY Buffalo, NY, USA; 4 Clebeland BioLabs, Inc, Buffalo, NY, USA; 5 INCURON, Inc. Moscow, Russia Summary Background: The Facilitates Chromatin Transcription (FACT) is involved in chromatin remodeling during transcription, replication and DNA repair and was considered to be ubiquitously expressed complex that had no known associations with any disease. However, we discovered that FACT is expressed in very limited number of cells of the adult mammalian organism, mostly presented by stem and undifferentiated cells (Garcia, 2011). Moreover a novel anti-cancer agents, Curaxins, entered recently Phase I clinical trials (lead curaxin, CBL0137), exert tumor cell killing though inhibition of FACT function (Gasparian, 2011). The goal of this study was to elucidate what role FACT plays in cancer. Material and methods: We assessed expression of FACT in normal and cancerous tissues of different organs on mRNA (>20,000 samples) and protein (>800 samples) levels to evaluate the correlation between FACT expression and clinical features of different cancers. We also ran in vitro and in vivo experiments to evaluate how modulation of FACT levels affects tumorigenic transformation and tumor cell properties. Finally we obtained genome wide distribution of FACT using ChIP-seq to identify genes which transcription requires FACT assistance. Results: FACT expression is significantly higher in tumors of patients with poor overall survival (all cancers, breast cancer (BC), NSCLC), higher incidence of metastasis (BC, NSCLC, RCC) and the presence of other markers associated with poor prognosis (BC, NSCLC, colon cancer). Ectopic expression of FACT in normal cells does not drive transformation, but increases efficiency of oncogene-driven transformation. Conversely a reduction of FACT level, using a RNAi approach, reduces transformation efficiency and interferes with tumor, but not normal cell growth. Genome wide analysis revealed non-random FACT distribution in tumor cells with significant enrichment over the bodies of genes regulated by transcriptional factors associated with cancer (Myc, AP1-, ets-families, YY1), stress response (NF-kB, HSF1, HIF1a) and pluripotent cell state (Oct3/4, Myc, Hox family). This pattern suggests selective assistance of FACT to the transcription of genes involved in cancer and early development. Conclusion: FACT is an attractive target and marker of poorly differentiated aggressive cancers based on its role as an accelerator of oncogenic transformation through selective chromatin remodeling of genes involved in cancer stress response and maintenance of pluripotent cell state. Background Curaxins , small molecule FACT inhibitors (Ref1) Lead FACT inhibitor,CBL0137 • Water soluble, orally bioavailable • Not a substrate of multidrug transporters • Penetrates blood brain barrier • Is in Phase I trial against advanced treatment resistant cancers and refractory lymphomas Toxicity to NCI60 panel CBL0137: broad anti-cancer activity in mouse models SSRP1 is a marker of poorly differentiated aggressive cancers (Ref.3) liver lung pancreas kidney cerebellum adrenals Breast adenocarcinoma Bladder Adenocarcinoma NSCLC Snapshot of FACT expression FACT expression in normal tissues (Ref.2) SSRP1 is not expressed in most of differentiated adult tissues (more details in Ref.2) FACT is an ”accelerator” of malignant transformation FACT dependent transcriptional programs Breast cancer • Curaxins were identified in the chemical screening of simultaneous activators of p53 and inhibitors of NF-kB in tumor cells • Effect of curaxins on p53, NF-kB and toxicity to tumor cells result from depletion of active FACT from tumor cells SSRP1-GFP SSRP1-GFP PK and tissue distribution Toxicity to tumor vs normal cells 0 2 4 6 8 10 12 14 0 5 10 25 30 35 Mean Fold Tumor Growth 0 2 4 6 8 10 12 14 0 5 10 25 30 35 Mean Fold Tumor Growth Vehicle Control 30 mg/kg CBL0137 40 mg/kg Sunitinib * * * * * * * 0 5 10 15 20 25 30 35 40 0 4 7 11 13 15 18 21 25 Mean Ffold Tumor Growth control CBL137 * * * * * Vehicle Control 30mg/kg CBL137 0 2 4 6 8 10 12 14 16 18 20 1 5 7 10 12 14 16 19 21 23 25 28 Days from start of treatment Mean Fold Tumor Growth Vehicle Control 30mg/kg CBL137 * * * * * * * * * * Days from start of treatment Days from start of treatment Days from start of treatment C CBLC137 CBLC137 0 2 4 6 8 10 12 14 16 0 10 20 Days from start of treatment Mean Fold Tumor Growth 0 2 4 0 10 30 ** * * * * Vehicle Control 30mg/kg CBL137 42mg/kg 5FU 50mg/kg Irinothecan 7.5mg/kg Oxaliplatin CBLC137 FACT is a marker of normal and cancer stem cells Intestine A - bone marrow; B - lymph node; C – mucosa associated lymphoid tissue; D- spleen A - ovary; B – infundibulum of fallopian tube; C – uterus. References 1. Gasparian AV, Burkhart CA, Purmal AA, Brodsky L, Pal M, Saranadasa M, Bosykh DA, Commane M, Guryanova OA, Pal S, Safina A, Sviridov S, Koman IE, Veith J, Komar AA, Gudkov AV, Gurova KV: Curaxins: Anticancer Compounds That Simultaneously Suppress NF-{kappa}B and Activate p53 by Targeting FACT. Sci Transl Med 2011, 3(95):95ra74. PMID: 21832239. 2. Garcia H, Fleyshman D, Kolesnikova K, Safina A, Commane M, Paszkiewicz G, Omelian A, Morrison C, Gurova KV: Expression of Facilitates Chromatin Transcription complex in mammalian tissues suggests FACT role in maintaining of undifferentiated state of cells. Oncotarget 2011, Oct 13. [Epub ahead of print] 3. Garcia H, Miecznikowski JC, Safina A, Commane M, Ruusulehto A, Kilpinen S, Leach RW, Attwood K, Yan Li, Degan S, Omilian AR, Guryanova O, Papantonopoulou O, Wang J, Buck M, Liu S, Morrison C, Gurova KV. FAcilitates Chromatin Transcription complex is an "accelerator" of tumor transformation and potential marker and target of aggressive cancers. Cell Reports, 2013. Published online July 3. Conclusions • FACT is a marker of aggressive poorly differentiated tumors • FACT is expressed at the highest levels in normal and cancer stem cells • Targeting FACT is a promising anti-cancer strategy • CBL0137 is the first FACT inhibitor in clinical trials Lung cancer Pancreatic ductal adenocarcinoma HMEC transformed in vitro (H-Ras, Myc, shp53,shP16) PDA patient’ transplants In vitro transformation of HMEC Genetic Chemical SSRP1 genome-wide distribution
Transcript of FACT (Facilitates chromatin transcription) complex as a ... · SPT16 SSRP1 b-actin FACT...
SPT16
SSRP1
b-actin
FACT (Facilitates chromatin transcription) complex as a marker and target of
aggressive poorly differentiated cancers Henry Garcia1, Alfiya Safina1, Daria Fleyshman1, Catherine Burkhart4,, Andrei Purmal5, Jeffrey C Miecznikowski3, Angela R. Omilian2, Carl Morrison2, Katerina V Gurova1
1Department of Cell Stress Biology, 2Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, 3Department of Biostatistics, SUNY Buffalo, NY, USA; 4Clebeland BioLabs, Inc, Buffalo, NY, USA; 5INCURON, Inc. Moscow, Russia
Summary Background: The Facilitates Chromatin Transcription (FACT) is involved in chromatin remodeling during transcription, replication and DNA repair and was considered to be ubiquitously expressed complex that had no known associations with any disease. However, we discovered that FACT is expressed in very limited number of cells of the adult mammalian organism, mostly presented by stem and undifferentiated cells (Garcia, 2011). Moreover a novel anti-cancer agents, Curaxins, entered recently Phase I clinical trials (lead curaxin, CBL0137), exert tumor cell killing though inhibition of FACT function (Gasparian, 2011). The goal of this study was to elucidate what role FACT plays in cancer. Material and methods: We assessed expression of FACT in normal and cancerous tissues of different organs on mRNA (>20,000 samples) and protein (>800 samples) levels to evaluate the correlation between FACT expression and clinical features of different cancers. We also ran in vitro and in vivo experiments to evaluate how modulation of FACT levels affects tumorigenic transformation and tumor cell properties. Finally we obtained genome wide distribution of FACT using ChIP-seq to identify genes which transcription requires FACT assistance. Results: FACT expression is significantly higher in tumors of patients with poor overall survival (all cancers, breast cancer (BC), NSCLC), higher incidence of metastasis (BC, NSCLC, RCC) and the presence of other markers associated with poor prognosis (BC, NSCLC, colon cancer). Ectopic expression of FACT in normal cells does not drive transformation, but increases efficiency of oncogene-driven transformation. Conversely a reduction of FACT level, using a RNAi approach, reduces transformation efficiency and interferes with tumor, but not normal cell growth. Genome wide analysis revealed non-random FACT distribution in tumor cells with significant enrichment over the bodies of genes regulated by transcriptional factors associated with cancer (Myc, AP1-, ets-families, YY1), stress response (NF-kB, HSF1, HIF1a) and pluripotent cell state (Oct3/4, Myc, Hox family). This pattern suggests selective assistance of FACT to the transcription of genes involved in cancer and early development. Conclusion: FACT is an attractive target and marker of poorly differentiated aggressive cancers based on its role as an accelerator of oncogenic transformation through selective chromatin remodeling of genes involved in cancer stress response and maintenance of pluripotent cell state. Background
Curaxins , small molecule FACT inhibitors (Ref1)
Lead FACT inhibitor,CBL0137
• Water soluble, orally bioavailable
• Not a substrate of multidrug
transporters
• Penetrates blood brain barrier
• Is in Phase I trial against advanced
treatment resistant cancers and
refractory lymphomas
Toxicity to NCI60 panel
CBL0137: broad anti-cancer activity in mouse models
SSRP1 is a marker of poorly differentiated
aggressive cancers (Ref.3)
liver lung pancreas
kidney cerebellum adrenals
Breast adenocarcinoma Bladder Adenocarcinoma NSCLC
Snapshot of FACT expression
FACT expression in normal tissues (Ref.2)
SSRP1 is not expressed in most of differentiated
adult tissues (more details in Ref.2)
FACT is an ”accelerator” of malignant
transformation
FACT dependent transcriptional programs
Breast cancer
• Curaxins were identified in the chemical screening of simultaneous activators of p53 and inhibitors of NF-kB in tumor cells
• Effect of curaxins on p53, NF-kB and toxicity to tumor cells result from depletion of active FACT from tumor cells
SSRP1-GFP SSRP1-GFP
PK and tissue distribution
Toxicity to tumor vs normal cells
CB
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CBLC137
D E
FACT is a marker of normal and cancer stem cells
Intestine
A - bone marrow; B - lymph node; C – mucosa associated lymphoid tissue; D- spleen
A - ovary; B – infundibulum of fallopian tube; C – uterus.
References 1. Gasparian AV, Burkhart CA, Purmal AA, Brodsky L, Pal M, Saranadasa M, Bosykh DA,
Commane M, Guryanova OA, Pal S, Safina A, Sviridov S, Koman IE, Veith J, Komar AA, Gudkov AV, Gurova KV: Curaxins: Anticancer Compounds That Simultaneously Suppress NF-{kappa}B and Activate p53 by Targeting FACT. Sci Transl Med 2011, 3(95):95ra74. PMID: 21832239.
2. Garcia H, Fleyshman D, Kolesnikova K, Safina A, Commane M, Paszkiewicz G, Omelian A, Morrison C, Gurova KV: Expression of Facilitates Chromatin Transcription complex in mammalian tissues suggests FACT role in maintaining of undifferentiated state of cells. Oncotarget 2011, Oct 13. [Epub ahead of print]
3. Garcia H, Miecznikowski JC, Safina A, Commane M, Ruusulehto A, Kilpinen
S, Leach RW, Attwood K, Yan Li, Degan S, Omilian AR, Guryanova O,
Papantonopoulou O, Wang J, Buck M, Liu S, Morrison C, Gurova KV.
FAcilitates Chromatin Transcription complex is an "accelerator" of tumor
transformation and potential marker and target of aggressive cancers. Cell
Reports, 2013. Published online July 3.
Conclusions • FACT is a marker of aggressive poorly
differentiated tumors • FACT is expressed at the highest levels in normal
and cancer stem cells • Targeting FACT is a promising anti-cancer strategy • CBL0137 is the first FACT inhibitor in clinical trials
Lung cancer
Pancreatic ductal adenocarcinoma
HMEC transformed in vitro (H-Ras, Myc, shp53,shP16)
PD
A p
atie
nt’
tra
nsp
lan
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In vitro transformation of HMEC
Gen
etic
C
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mic
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SSRP1 genome-wide distribution
