Lysosomal transmembrane protein TMEM106B alters TFEB signaling and the tumor immune microenvironment Amanda Warner, Samrat T. Kundu, Rakhee Bajaj, Bertha L. Rodriguez, Don L. Gibbons University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences Department of Thoracic Head & Neck Medical Oncology; Contact info: awarner2@mdanderson.org Introduction A
Genes (FC<-1, p<0.05) in 344SQ TMEM sh2 vs. Scr subQ tumors
Genes (FC>1, p<0.05) in 393P TMEM vs. mCherry subQ tumors
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Non-small cell lung cancer (NSCLC) is the leading cause of cancer related deaths particularly due to its high propensity to metastasize 1 . Our lab performed an in vivo screen 2 and identified transmembrane lysosomal protein 106B, TMEM106B, as a novel driver of NSCLC metastasis. TMEM106B activates transcription factor EB (TFEB) and lysosomal exocytosis which releases cathepsins into the tumor microenvironment (TME) that promote metastasis 3 . Beyond cathepsin release, the mechanism of how TMEM106B alters cell biology and the TME to promote metastasis remains unclear. TFEB is necessary for lysosomal exocytosis 4 , autophagy 4 , invasion in prostate cancer 5 , and activates immune gene transcription 6,7 . We hypothesize that (1) TMEM106B mediated TFEB activation upregulates autophagy to promote metastasis and (2) TMEM106B is dependent on TFEB to regulate lysosomal exocytosis and secretion in NSCLC which alter the tumor immune microenvironment (TIME) to become pro-metastatic . It will be crucial to know how TMEM106B mechanistically alters cellular biology and the TIME as this will provide new therapeutic targets and strategies to limit NSCLC metastasis. A B
IPA Canonical Pathway Analysis: -ERK/MAPK -HER2 signaling -Autophagy -ER signaling
Figure 6: Autophagy predicted to be dependent on TMEM106B in murine tumors. Canonical Pathways by Ingenuity Pathway Analysis (IPA) on microarray. Venn Diagram by Venny2.1, FC=Fold Change.
*Lower GFP exposure for 344SQ-GFP
B
C
393P-GFP-TMEM +TFEB-mCherry
393P-GFP +TFEB-mCherry
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2.0 90 100
*
*
*
1.5
1.0
0.5
TMEM106B HEXA NEU1 CTSD 0.0
344SQ-Vec
344SQ-TMEM106B
Figure 3: TMEM106B induces lysosomal biogenesis and TFEB activation. (A) Lysotracker dye (RFP) identifying lysosomes. (B) TMEM106B enhances TFEB nuclear translocation 3 and (C) elevates TFEB target gene expression.
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p <0.001
C
B.1
A
75
SubQ Tumor p =0.02
200
70
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Conclusions • TMEM106B is necessary for NSCLC invasion and metastasis. • TMEM106B induces TFEB activation which enhances lysosomal biogenesis but not autophagosome number or flux in vitro . • TMEM106B tumors have less total CD8+ T-cells, more exhausted T-cells, and altered myeloid cell populations suggesting TMEM106B could contribute to immunosuppression. Figure 7: TMEM106B is not sufficient to alter autophagy flux. Autophagy flux measured by starving cells in HBSS +/- BafilomycinA1, which inhibits lysosome acidification and autophagosome degradation, and measuring LC3-II and p62, each normalized to actin.
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Vec TMEM
mCherry TMEM106B
B.2
CD8+ IHC
Figure 1: TMEM106B identified as a metastasis driver in NSCLC from in vivo murine screen. (A) Cell lines generated from KP murine GEM tumors. (B) Gene list for in vivo screen 2 . (C) TMEM106B was present in >2 lung metastasis lesions per five individual mice 2 . Results
393P: mCherry
TMEM106B
Figure 4: TMEM106B predicted to affect transcriptional regulation of immune signaling genes. (A) Ingenuity Pathway Analysis (IPA) on genes (FC>1, p<0.05) from microarray on TMEM106B subQ tumors which (B) have less CD8+ T-cells by IHC. (C) TMEM106B enhances surface PD-L1 upon co-culture with activated naïve immune cells.
Future Directions
• Perform qPCR immune gene screen and cytokine array to validate immune genes/signaling pathways regulated by TMEM106B as identified by microarray. • Determine contribution of TFEB as a downstream target of TMEM106B mediated alterations on TIME to affect metastasis (Image by BioRender).
B
D
A
p <0.005
1.5
TMEM106B
344SQ
p <0.05
Scr sh1 sh2
C
A
1.0
Exhausted CD8 + T Cells 0.0555
400
Effector Memory CD8+ T Cells 0.0121
52kDa
Naive CD8+ T Cells
CD8+ T Cells 0.1669
40
1
0.81 0.36
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Vec TMEM106B
300
0.5
15
15
0.0004
80
30
200
10
10
60
0.0
20
Scr sh1 sh2
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C
p =0.006
100
TMEM106B
5
5
*
10
20
p =0.002
344SQ Invasion
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0
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0
0
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Vec TMEM
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MDSC 0.0195
150
p< 0.0001
Macrophage <0.0001
M2 Macrophage 0.0002
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100
25
100
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20
4
60
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15
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40
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0
20
References and Funding
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0
Scr sh1 sh2
1 2 3 4 1 2 3 4
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Vec
TMEM106B
TMEM106B
Vec TMEM
Vec TMEM
Vec TMEM
344SQ SubQ Tumors
1) Siegel, R. L., et al., CA Cancer J Clin. , 2022 2) Grzeskowiak, C. L., et al., Nat. Commun ., 2018 3) Kundu, S. T., et al., Nat. Commun ., 2018 4) Medina, D. L., et al., DevCell. , 2011 5) Zhu, X., et al., Front. Oncol., 2021 6) El-Houjeiri, L., et al., Cell Rep. , 2019 7) Palmieri, M., et al., Hum Mol Genet. , 2011
344SQ
This work was funded by D.L.G. grants: NIH R37 (CA214609); CPRIT MIRA(RP160562-P3); SPORE (2P50CA070907-21A).
Figure 5: TMEM106B overexpression alters the TIME to be immunosuppressive. (A) subQ tumor volume unchanged. (B) Tmem106B tumor RNA expression. (C) T- lymphocyte and (D) myeloid population percentage changes within 344SQ tumors.
Figure 2: TMEM106B is necessary for invasion and metastasis. (A, B) Knock-down of TMEM106B by RNA and protein. (C) TMEM106B is necessary for invasion in vitro . (D) TMEM106B is necessary for murine NSCLC metastasis to lungs 3 .
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