Supplementary Figure 1 A Cisplatin Cisplatin Cisplatin Late resistance Sensitive cells Early resistance (H357CisR8M) (H357CisS) (H357CisR4M) B SCC-4 CisS SCC9 CisS H357 CisS SCC-4 CisR 4M SCC9 CisR 4M 120 120 H357 CisR 4M SCC4 CisR 8M * H357 CisR 8M 120 SCC9 CisR 8M 100 * * * * 100 * * * * * 100 * * * * * * * * * * * * * * 80 * 80 * 80 60 60 60 40 40 40 % of cell viability of cell % % of cell viability of cell % 20 ofviability cell % 20 20 0 0 0 5 1 5 10 15 20 25 30 35 40 1 5 2.5 7.5 10 15 20 25 30 35 40 2.5 7.5 2.5 7.5 10 15 20 25 30 35 12.5 12.5 DMSO DMSO DMSO Cisplatin (M) Cisplatin (M) Cisplatin (M) C 100 100 80 80 60 60 40 A375 CisS 40 A549 CisS A549 CisR A375 CisR % of cell viability of cell % 20 % ofviability cell % 20 0 0 1 2 3 4 5 6 8 10 12 14 16 18 20 25 30 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 DMSO Cisplatin (M) DMSO Cisplatin (M) Supplementary figure 1: Characterization of sensitive, early and late cisplatin resistant lines: A) Schematic presentation of establishing sensitive, early and late cisplatin resistant cancer lines B) Sensitive, early and late cisplatin resistant pattern (CisS, CisR4M and CisR8M) of H357, SCC9 and SCC4 cells were treated with indicated concentrations of cisplatin for 48h and cell viability was determined by MTT assay (n=3, *: P < 0.05). C) Sensitive and late cisplatin resistant lung cancer (A549) and melanoma (A375) lines were established as described in method section. Sensitive and resistant cells were treated with cisplatin with indicated concentrations for 48h and cell viability was determined by MTT assay (n=3 *: P < 0.05). Supplementary Figure 2 4R2 4R11 8R12 8R11 8R2 4R12 Supplementary figure 2: Global proteomic profiling of sensitive, early and late chemoresistant cells. The lysates were isolated from parental sensitive (H357CisS), early (H357CisR4M) and late (H357CisR8M) cisplatin resistant cells and subjected to global proteomic profiling. The dendrogram represents the dysregulated genes from proteomic analysis between early (H357CisR4M) and late (H357CisR8M) cisplatin resistant cells after normalizing with parental sensitive (H357CisS) cells. Supplementary Figure 3 B C A Chemotherapy Chemotherapy * 300 responder non-responders 200 A375CisR H357CisR A549CisS A375CisS A549CisR H357CisS 100 37 kD PDL-1 Q- Score ofScorePDL-1 Q- 1 10.25 1 2.03 1 2.34 0 Chemotherapy Chemotherapy 42 kD β-Actin responder non-responder (n=11) (n=23) D E F Paired Samples After Chemotherapy 300 P< 0.0001 Before Chemotherapy Before Chemotherapy r = 0.7228 400 After Chemotherapy 1 - 200 L 300 D P f PDL-1 o 200 e 100 r o c S - 100 Q 0 0 0 50 100 150 200 250 Pt1 Pt2 Pt3 Pt4 Pt5 CMTM6 G HNSCC RFS plot ESCC RFS plot LUSC RFS plot Supplementary figure 3: PDL1 expression is elevated in cisplatin resistant carcinomas: A) Cell lysates from indicated cisplatin resistant and sensitive cancer lines were isolated and subjected to immunoblotting (n=3) against PDL1 and β-actin antibodies. B) Protein expression of PDL1 was analyzed by IHC in chemotherapy-responder and chemotherapy-non-responder OSCC tumors C) IHC scoring of PDL1 (Median, n=11 for chemotherapy-responder and n=23 for chemotherapy-non-responder) *: P < 0.05. D) Protein expression of PDL1 was analyzed by immunohistochemistry (IHC) in pre- and post-TPF treated paired tumor samples for chemotherapy-non-responder patients E) IHC scoring of CMTM6 (n=6) F) Co-relation analysis of CMTM6 and PDL1 in chemotherapy-non-responder patients (n=23) G) Relapse Free Survival (RFS) plot for PDL1 using KM-Plotter. *For IHC scoring, Q Score =Staining Intensity × % of IHC Staining. Supplementary Figure 4 A 100 100 y 80 y 80 t t i i l l i i b 60 b 60 a a i i v v l l l l 40 40 e e c c % % 20 20 0 0 Cisplatin (μM) UT 2.5 5 10 UT 2.5 5 10 UT 2.5 5 10 UT 2.5 5 10 CMTM6 OE - - - - + + + + - - - - + + + + H357 SCC9 50 60 B 50 40 h h t t a 40 a e e 30 d d l l l l 30 e e 20 C C 20 % % 10 10 0 0 Cisplatin (μM) UT 5 10 UT 5 10 UT 5 10 UT 5 10 CMTM6 OE - - - + + + - - - + + + H357 SCC9 Supplementary figure 4: Ectopic overexpression of CMTM6 induced drug resistance phenotype in Cisplatin sensitive cells: A) CMTM6 was overexpressed in cisplatin sensitive H357 and SCC9 cell lines and treated with cisplatin with indicated concentration for 48h. Cell viability was determined by MTT (n=3), *: P < 0.05 by B) Cells were treated as described in panel E and cell death was determined by annexin V/7AAD assay using flow cytometer (n=3). Supplementary Figure 5 A H357 CisR SCC9 CisR 100 100 y 80 y 80 t t i i l l i i b b a 60 a 60 i i v v l l l l e e 40 40 c c % % 20 20 0 0 Cisplatin (μM) 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 NT shRNA + + + + - - - - + + + + - - - - ENO1 shRNA - - - - + + + + - - - - + + + + B H357 CisR SCC9 CisR 60 80 h h 60 t t a a 40 e e d d l l l l 40 e e C C 20 % % 20 0 0 Cisplatin (μM) 0 5 10 0 5 10 0 5 10 0 5 10 NT Sh RNA + + + - - - + + + - - - ENO1 Sh RNA - - - + + + - - - + + + Supplementary figure 5: ENO1 knockdown re-sensitises chemoresistant cells to cisplatin : A) Cisplatin resistant H357 and SCC9 cells stably expressing NTShRNA and ENO1ShRNA were treated with cisplatin for 48h and cell viability was determined by MTT assay B) Cisplatin resistant cells stably expressing NTShRNA and ENO1ShRNA were treated with cisplatin for 48h and after which cell death was determined by annexin V/7AAD assay using flow cytometer. Supplementary Figure 6 Nt Sh UT Vs Nt Sh Cisplatin CMTM6 Sh UT Vs CMTM6 Sh Cisplatin Supplementary figure 6: Pathway enrichment analysis: Pathway enrichment analysis of NT Sh UT vs NT Sh Cisplatin, CMTM6 Sh UT vs CMTM6 Sh Cisplatin Supplementary Figure 7 A NtSh UT Vs CMTM6Sh UT C NtSh UT Vs CMTM6Sh UT B NtSh Cisplatin Vs CMTM6Sh Cisplatin D NtSh Cisplatin Vs CMTM6Sh Cisplatin Supplementary figure 7: Wnt target genes are deregulated in CMTM6 dependent manner: A, B) Heat map of WNT signaling gene dysregulated in NT Sh UT vs CMTM6 Sh UT, NT Sh Cisplatin treated vs CMTM6 Sh Cisplatin treated. C, D) Network analysis of WNT pathway genes in NtSh Vs CMTM6Sh UT and NtSh Vs CMTM6Sh Cisplatin. Supplementary Figure 8 A B β-Catenin SCC9CisR 1 0.17 0.47 1 0.16 0.44 1 0.15 0.41 1 0.19 0.47 NtshRNA CMTM6 1 0.03 0.07 1 0.06 0.04 1 0.02 0.05 1 0.08 0.01 SCC9CisR β-Actin CMTM6KD NT sh RNA + - - + - - + - - + - - - + + - + + Non-phospho DAPI MERGE CMTM6 sh RNA - + + - + + β-Catenin Cisplatin (10 μM) - - - + + + - - - + + + LiCl (20mM for 24 h) - - + - - + - - + - - + SCC9CisR H357CisR C n n n o 1.5 o o i 1.5 1.5 i NT ShRNA NT ShRNA i NT ShRNA s s s s s s e e e r CMTM6 ShRNA#1 CMTM6 ShRNA#1 CMTM6 ShRNA#1 r r p p p x x x e e 1.0 e 1.0 1.0 A A A N N N R R R m m m 0.5 0.5 0.5 e e e v v v i i i t t t a a a l l l e e e R R 0.0 0.0 R 0.0 CD44 c-Myc CCND1 TCF4 CD44 c-Myc CCND1 TCF4 CD44 c-Myc CCND1 TCF4 H357CisR SCC9CisR PDC1 H357CisR CMTM6 KD D SCC9CisR CMTM6 KD H357CisR CMTM6 KD + PCMV6 CMTM6 SCC9CisR CMTM6 KD + PCMV6 CMTM6 n n 6 4 o o i i s s s s e e r r p p 3 x x 4 e e A A N N 2 R R m m 2 e e 1 v v i i t t a a l l e e 0 0 R R CD44 C-MYC CCND1 TCF4 LEF1 CD44 C-MYC CCND1 TCF4 LEF1 Supplementary figure 8: CMTM6 correlates with Wnt signalling : A) Confocal imaging was done with the indicated antibodies B) Lysates were isolated from indicated lines and immunoblotting (n=3) was performed with the indicated antibodies C) Relative mRNA (fold change) expression of indicated genes were analyzed by qRT PCR in indicted cells stably expressing NTShRNA or CMTM6ShRNA#1 (mean ±SEM, n=3). D) CMTM6 was overexpressed in chemoresistant CMTM6KD (ShRNA#2) and relative mRNA expression of indicated genes were determined by qRT PCR (mean ±SEM, n=3). Supplementary Figure 9 p-value = p-value p-value p-value = 0 = 4.7e- =2.4e- 0 R = 0.41 10 08 R = 0.36 R = 0.27 R = 0.24 (CD44 (CD44 TPM) (TCF4 (TCF4 TPM) 2 2 (MMP14 (MMP14 TPM) (LEF1 (LEF1 TPM) 2 2 log log log log log2(CMTM6 TPM) log2(CMTM6 TPM) log2(CMTM6 TPM) log2(CMTM6 TPM) p-value = 0 p-value = p-value = p-value = R = 0.38 6e-06 0 8.7e-14 R = 0.2 R = 0.41 R = 0.32 (ID2 (ID2 TPM) (ENC1 (ENC1 TPM) 2 2 (PPARA TPM) 2 (JAG1 (JAG1 TPM) 2 log log log log log (CMTM6 TPM) log2(CMTM6 TPM) log (CMTM6 TPM) 2 2 log2(CMTM6 TPM) Supplementary figure 9: Expression correlation between CMTM6 and Wnt target prosurvival genes : TCF4, LEF1, CD44, MMP14, ENC1, ID2, PPARA and JAG1 RNA expression in the TCGA HNSCC database.