C57BL/6 mice, B6.Ly5.1, B6.129P2(C)-Ccr7tm1Rfor/J (CCR7-deficient), and NSG mice were from Jackson Laboratories (Bar Harbor, ME)1. B6.CARMA1-deficient mice were gift from Dr. Marisa Alegre (University of Chicago). All mice were bred and/or maintained in a specific pathogen-free conditions (University of New Mexico) and conform to the principles outlined by the Animal Welfare Act and NIH guidelines, IACUC protocol 15-101294. All efforts were made to minimize suffering.
CEM (Human T-Acute lymphoblastic leukemia cell line), CEM- luciferase-GFP, CARMA1 knockdown CEM-luciferase-GFP were confirmed by cell line testing through ATCC.
Antibodies and reagents:
IRDye® 700 NFkB Consensus Oligonucleotide (829-07924) (Licor). CARD11 MISSION shRNA (SHCLNV-NM_032415) and PKCθ MISSION shRNA (SHCLNV-NM_006257) (Sigma). Human CD45-APC (2D1), CCR7-PE (3D12), CD45.1-PE-Cy7 (A20), CD45.2-APC (104), CD3-eFluor660 (17A2) CD4-PE (GK1.5), CD8-APC (53-6.7) (eBioscience, San Diego, CA). Antibodies: CARMA1 (N20), Bcl10 (H-197, 331.3), and IB (C-15) from Santa Cruz Biotech. Carma1 (1D12), p-CARMA1 (Ser652), p-ERK (Thr202/Tyr204), ERK (137F45) and Cell lysis buffer (9803) from Cell signaling. Human PKCθ (27) from BD Biosciences. CARMA1-GFP plasmid (RG222740) was from OriGene (Rockville, MD, USA). CCR7-mCherry plasmid was a gift from Dr. Vicente Planelles (University of Utah School of Medicine)2.
Retroviral transfection and bone marrow transplantation:
For knockdown of CEM cells: CEM cells were infected with lentivirus encoding firefly luciferase and GFP (gift from Stephan A. Grupp, MD, PhD, University of Pennsylvania)3. For mouse T-ALL model: The retroviral vector MigR1-Notch1∆E-GFP (gift from Dr. Adolfo A. Ferrnado, Columbia University, New York)4 was transfected into AmphoPhoenix5 cells, supernatants were collected and used after 36hrs. Bone marrow transplantation was done as described6. Engraftment of leukemic cells was analyzed 3-4 weeks after transfer.
Xenografted mice were injected intraperitoneally with 150mg/kg of luciferin and bioluminescence signals were monitored using the IVIS system 100 (Xenogen Corp.).
Transwell Migration Assay:
CEM-luciferase-GFP or CARMA1KD-luciferase-GFP were combined in a 1:1 ratio with non-fluorescent parental-CEM cells and added to the top of a Costar (Corning Acton, MA) 5.0 µm Transwell apparatus. 300ng/ml CCL21 was added to the bottom of the transwell apparatus or coated with 6µg/ml ICAM and cells were added to the top. Migrated cells were collected after 1-2 hours and non-GFP (parental CEMs) and GFP+ (parental CEMs or CARMA1KD-CEMs) were analyzed by flow cytometry. The ratio of parental CEMs:CARMA1KD-GFP+CEMs were calculated and then normalized to parental CEMs:parental-GFP+CEMs.
Histology was performed as described in7. Briefly, murine heads were stripped of soft tissues, fixed in 10% Neutral Buffered Formalin (CellPath, Powys, UK) and decalcified in Hilleman and Lee EDTA solution, paraffin embedded, stained using a Dako Autostainer with DAKO anti-Human CD45 (2B11+PD7/26), and viewed on an Axiostar plus microscope. Quantification of CNS infiltration was performed using a Hamamatsu Nanozoomer Digital Pathology slide scanner with digital slide management/image analysis software from Slidepath (Dublin).
Colocalization of CARMA1-GFP and CCR7-mCherry was done using the Amnis Imagestream (EMD Millipore, Seattle WA). Cells were transfected, stimulated, fixed with 4% PFA, and data captured with the Amnis ImagestreamX® MKII. Single cells were distinguished using brightfield imagery and quantitation of colocalization determined using Pearson’s coefficient using the Brightness Detail Similarity.
All statistics are described in individual figure legends with the statistical test used and exact p value reported. For comparisons of survival the Log-rank Mantel-Cox test was used. For comparisons of cell numbers in tissues, the Student’s t-test was used. For microarray data, the non-parametric Mann-Whitney test was used.
Microarray data analysis:
Microarray data from Figure 1C was from Gene Expression Omnibus (GEO) under Accession #: GSE132048. CARMA1 probes were compared between healthy bone marrow (n=74) versus T-ALL patient samples (n=174) and processed as done in9. Data for Figure 1D were taken from the Cancer Cell Line Encyclopedia (CCLE) project10 using the GEO Accession #: GSE36133 represented with RMA log2. Figure 2C was from Study 9404 11 GEO accession # GSE1461512. Non-CNS was CNS1 while the CNS group included CNS2 and CNS3. All statistics for microarray analyses were done using the Mann-Whitney-Wilcoxon (non-parametric) test with exact p-values as indicated using the statistical package R.
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2. Ramirez PW, Famiglietti M, Sowrirajan B, DePaula-Silva AB, Rodesch C, Barker E, et al. Downmodulation of CCR7 by HIV-1 Vpu results in impaired migration and chemotactic signaling within CD4(+) T cells. Cell Rep 2014 Jun 26; 7(6): 2019-2030.
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4. Herranz D, Ambesi-Impiombato A, Palomero T, Schnell SA, Belver L, Wendorff AA, et al. A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia. Nat Med 2014 Oct; 20(10): 1130-1137.
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Supplemental Figure Legends
Supplemental Figure 1: CARMA1 does not regulate basal proliferation or apoptosis of CEM cells. (A) Parental and CARMA1KD CEM cells were assayed for CARMA1 mRNA (left) and protein (right). (B,C) Parental and CARMA1KD CEM cells were assayed for (B) proliferation by cell counting over 4 days, (C) apoptosis by Annexin V and DAPI staining. Quantification on (C) shown in right panel was done by enumerating Annexin V+DAPI+ population and shows an average of 3 replicates.
Supplemental Figure 2: CARMA1 expression increases T-ALL tumor burden and T-ALL cell accumulation in specific organs. NSG animals xenografted with parental or CARMA1KD animals. (A, B) Xenografted animals were injected with luciferin and imaged. (A) Representative images of luciferase-positive parental and CARMA1KD CEM cells at week 2. (B) Quantitation of signal intensity (n=5 animals). P-values determined using 2-way ANOVA. (C) Lin- cells from bone marrow of C57Bl/6 and CARMA1-/- were transfected with Notch1∆E-GFP and transferred into irradiated B6.LY5.1 animals. Organs specified were removed on day 35 post cell transfer and GFP+LY5.2+ cells enumerated from WT/WT Notch1∆E (WT-NOTCH1-∆E) and WT/CARMA1-/- Notch1∆E (CARMA1KO-NOTCH1-∆E) animals. P-values shown used the Student’s t-test.
Supplemental Figure 3: CARMA1 regulates T-ALL cell migration in vivo to the CNS. Heads of WT/WT Notch1∆E (WT-NOTCH1-∆E) or WT/CARMA1-/- Notch1∆E(CARMA1KO-NOTCH1-∆E) were processed for H&E (in Methods). Images of sections through skull and meninges of two representative mice (of 4) and stained for H&E (left hand panel) or anti-GFP (right hand panel). Meningeal infiltration indicated by thick arrow and bone marrow by *. Scale bar represents 100µm.
Supplemental Figure 4: CARMA1 and CCR7-deficiency prolong survival in a mouse model of T-ALL similarly. Lin- cells were purified from bone marrow of WT C57Bl/6, CARMA1-/-, or CCR7-/- animals, transfected with Notch1∆E-GFP, and transferred into lethally irradiated B6.LY5.1 animals. Survival curves for WT/WT Notch1∆E (WT; n=12), WT/CARMA1-/- Notch1∆E (CARMA1KO; n=11), or WT/CCR7-/- Notch1∆E (CCR7KO; n=10) animals are shown. P-values between survival curves was determined using the Mantel-Cox test.
Supplemental Figure 5: CARMA1 colocalizes with CCR7. Parental CEM cells were transfected with CARMA1-GFP and CCR7-mCherry and imaged. Scale bar = 7µm.
Supplemental Figure 6: PKCθ expression does not affect T-ALL disease in a xenograft model. (A) NSG recipients were xenografted with parental CEM or PKCθKD CEM cells. Survival curves of NSG mice receiving parental or PKCθKD CEM cells. Parental CEM: n=22; PKCθKD CEM: n=6. Comparison of survival curves showed p>0.05 was determined using the Mantel-Cox test. (B) Luciferase-positive parental and PKCθKD CEM cells at week 2 and 3 post cell transfer with quantitation of signal intensity (n=5 for each). The Student’s t-test showed p>0.05 when comparing intensity curves.
Supplemental Table 1: CARMA1 regulates T-ALL cell accumulation in the CNS. (A) H&E sections from NSG mice xenografted with parental or CARMA1KD CEM cells were processed at indicated times and data for 2 animals shown. (B) H&E sections from B6.LY5.1 animals receiving WT Notch1∆E-GFP or CARMA1KO Notch1∆E-GFP transduced cells at week 5 post transfer and processed as described and analyzed for 4 animals shown.