#METABOLOMICS WORKBENCH juliehaines_20230509_073244 DATATRACK_ID:4015 STUDY_ID:ST002712 ANALYSIS_ID:AN004396 PROJECT_ID:PR001681
VERSION             	1
CREATED_ON             	May 25, 2023, 12:37 pm
#PROJECT
PR:PROJECT_TITLE                 	Ranolazine induced metabolic rewiring improves melanoma responses to targeted
PR:PROJECT_TITLE                 	therapy and immunotherapy
PR:PROJECT_SUMMARY               	Metabolic rewiring affects resistance of melanoma to targeted- and
PR:PROJECT_SUMMARY               	immuno-therapy. We have found that increased fatty acid oxidation (FAO) during
PR:PROJECT_SUMMARY               	late stages of BRAF inhibitor (BRAFi) treatment enables the establishment of
PR:PROJECT_SUMMARY               	acquired resistance. Targeting FAO with ranolazine in vivo once acquired
PR:PROJECT_SUMMARY               	BRAFi-resistance emerges delays tumour recurrence. Single cell RNAseq analysis
PR:PROJECT_SUMMARY               	revealed that ranolazine diminishes the transcriptional NGFRhigh neural crest
PR:PROJECT_SUMMARY               	stem cell subpopulation, which is refractory against BRAFi and immunotherapy.
PR:PROJECT_SUMMARY               	Moreover, by rewiring the methionine salvage pathway, ranolazine enhanced
PR:PROJECT_SUMMARY               	melanoma immunogenicity through increased antigen presentation and interferon
PR:PROJECT_SUMMARY               	signalling. Combination of ranolazine with anti-PD-L1 antibodies strongly
PR:PROJECT_SUMMARY               	improved survival in mice, where it increased lymphocyte infiltration and
PR:PROJECT_SUMMARY               	enhanced anti-tumour responses. Altogether, we show that ranolazine increases
PR:PROJECT_SUMMARY               	the efficacy of targeted melanoma therapy through fatty acid and methionine
PR:PROJECT_SUMMARY               	salvage metabolic rewiring. Importantly, our study suggests that ranolazine
PR:PROJECT_SUMMARY               	could sensitize BRAFi-resistant tumours to immunotherapy, by modulating melanoma
PR:PROJECT_SUMMARY               	cell recognition and immune infiltration. Ranolazine is an FDA and EMA-approved
PR:PROJECT_SUMMARY               	anti-anginal drug with very mild side effects, and our preclinical data
PR:PROJECT_SUMMARY               	encourage its use as a therapeutic option to improve the two main therapeutic
PR:PROJECT_SUMMARY               	strategies currently used to treat metastatic melanoma.
PR:INSTITUTE                     	University of Colorado Denver
PR:LABORATORY                    	Lab of Angelo D'Alessandro in collaboration with lab of Imanol Arozarena
PR:LAST_NAME                     	Haines
PR:FIRST_NAME                    	Julie
PR:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
PR:EMAIL                         	julie.haines@cuanschutz.edu
PR:PHONE                         	3037243339
#STUDY
ST:STUDY_TITLE                   	Ranolazine induced metabolic rewiring improves melanoma responses to targeted
ST:STUDY_TITLE                   	therapy and immunotherapy - metabolomics
ST:STUDY_SUMMARY                 	Metabolomics analysis was performed on A375 melanoma cells resistant to BRAF
ST:STUDY_SUMMARY                 	inhibitor vemurafenib (VR) and cells resistant to VR and treated with fatty acid
ST:STUDY_SUMMARY                 	oxidation inhibitor ranolazine.
ST:INSTITUTE                     	University of Colorado Denver
ST:LAST_NAME                     	Haines
ST:FIRST_NAME                    	Julie
ST:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
ST:EMAIL                         	julie.haines@cuanschutz.edu
ST:PHONE                         	3037243339
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:GENDER                        	Female
SU:CELL_BIOSOURCE_OR_SUPPLIER    	ATCC
SU:CELL_STRAIN_DETAILS           	A375
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	A375P 5	Treatment:VEHICLE	RAW_FILE_NAME=AG-M-1
SUBJECT_SAMPLE_FACTORS           	-	A375P 6	Treatment:VEHICLE	RAW_FILE_NAME=AG-M-2
SUBJECT_SAMPLE_FACTORS           	-	A375P 7	Treatment:VEHICLE	RAW_FILE_NAME=AG-M-3
SUBJECT_SAMPLE_FACTORS           	-	A375P 8	Treatment:VEHICLE	RAW_FILE_NAME=AG-M-4
SUBJECT_SAMPLE_FACTORS           	-	A375 VR 5	Treatment:VEMURA	RAW_FILE_NAME=AG-M-5
SUBJECT_SAMPLE_FACTORS           	-	A375 VR 6	Treatment:VEMURA	RAW_FILE_NAME=AG-M-6
SUBJECT_SAMPLE_FACTORS           	-	A375 VR 7	Treatment:VEMURA	RAW_FILE_NAME=AG-M-7
SUBJECT_SAMPLE_FACTORS           	-	A375 VR 8	Treatment:VEMURA	RAW_FILE_NAME=AG-M-8
SUBJECT_SAMPLE_FACTORS           	-	A375 VR-Rano 5	Treatment:VEMURA+RANO	RAW_FILE_NAME=AG-M-9
SUBJECT_SAMPLE_FACTORS           	-	A375 VR-Rano 6	Treatment:VEMURA+RANO	RAW_FILE_NAME=AG-M-10
SUBJECT_SAMPLE_FACTORS           	-	A375 VR-Rano 7	Treatment:VEMURA+RANO	RAW_FILE_NAME=AG-M-11
SUBJECT_SAMPLE_FACTORS           	-	A375 VR-Rano 8	Treatment:VEMURA+RANO	RAW_FILE_NAME=AG-M-12
#COLLECTION
CO:COLLECTION_SUMMARY            	All cell lines had been authenticated in 2021. Cells were expanded to generate
CO:COLLECTION_SUMMARY            	enough vials from a single batch before the start of the study. Cell lines were
CO:COLLECTION_SUMMARY            	cultured in Dulbecco’s Eagle’s Medium (DMEM) (cat#61965026, Gibco)
CO:COLLECTION_SUMMARY            	supplemented with 10% fetal bovine serum (cat#10500064, Gibco) plus 1%
CO:COLLECTION_SUMMARY            	penicillin/streptomycin (cat#15140122, Gibco). Cells were grown at 37°C in a 5%
CO:COLLECTION_SUMMARY            	CO2 environment.
CO:SAMPLE_TYPE                   	Melanoma cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Melanoma cells seeded in 6-well plates were treated for 7 days with a high dose
TR:TREATMENT_SUMMARY             	(5 or 10μM as indicated) of vemurafenib before switching to a lower
TR:TREATMENT_SUMMARY             	concentration of the drug (0,5 or 1μM). Fresh medium and vemurafenib was added
TR:TREATMENT_SUMMARY             	once weekly for further 3-4 weeks until arising colonies grew to confluence.
TR:TREATMENT_SUMMARY             	Fatty Acid oxidation inhibitors ranolazine, etomoxir or thiroridazine were added
TR:TREATMENT_SUMMARY             	once a week.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites from frozen cell pellets were extracted with cold 5:3:2
SP:SAMPLEPREP_SUMMARY            	MeOH:acetonitrile:water at a concentration of 2 million cells/mL. Samples were
SP:SAMPLEPREP_SUMMARY            	vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10
SP:SAMPLEPREP_SUMMARY            	min, 10,000 g, 4 degrees C) and transferred to autosampler vials.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Positive C18
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile 0.1% formic acid
CH:FLOW_GRADIENT                 	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min
CH:FLOW_GRADIENT                 	95-5% B, 3-5 min hold at 5% B
CH:FLOW_RATE                     	450 uL/min
CH:COLUMN_TEMPERATURE            	45
CH:SAMPLE_INJECTION              	10 uL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 480
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	We use a Thermo Orbitrap Exploris 120 (not available in the drop down box).
MS:MS_COMMENTS                   	Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms,
MS:MS_COMMENTS                   	microscans 1, automatic gain control (AGC) detection duration 20 msec, source
MS:MS_COMMENTS                   	voltage 3.5 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath
MS:MS_COMMENTS                   	gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to
MS:MS_COMMENTS                   	mzXML using RawConverter. Metabolites were annotated and integrated using Maven
MS:MS_COMMENTS                   	in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	A375P 5	A375P 6	A375P 7	A375P 8	A375 VR 5	A375 VR 6	A375 VR 7	A375 VR 8	A375 VR-Rano 5	A375 VR-Rano 6	A375 VR-Rano 7	A375 VR-Rano 8
Factors	Treatment:VEHICLE	Treatment:VEHICLE	Treatment:VEHICLE	Treatment:VEHICLE	Treatment:VEMURA	Treatment:VEMURA	Treatment:VEMURA	Treatment:VEMURA	Treatment:VEMURA+RANO	Treatment:VEMURA+RANO	Treatment:VEMURA+RANO	Treatment:VEMURA+RANO
L-alanine	1873299	1839249	960536	1449300	1017454	761078	582263	750160	605128	489356	522599	689910
L-arginine	790670	875694	790815	841990	1405993	1232484	1438302	1696229	1057014	1522084	813558	1535090
L-asparagine	15533	14431	19767	28455	193050	215190	241579	305134	103194	80326	74205	127645
L-aspartate	172475	192749	185383	193803	410353	369211	419312	480910	231425	336655	226576	380082
L-cysteine	13152	37748	25093	25438	20824	8896	14525	12593	8777	20522	6401	17866
L-glutamate	7923250	7960536	6027780	8106199	4435238	3718165	3283682	4156250	2641219	2856216	2281330	3149223
L-glutamine	935982	944938	647313	914996	682121	683041	510702	554099	207697	180960	190611	274676
L-leucine/isoleucine	28326898	27728438	16190471	22520454	10938471	7327908	6561877	8064346	4868628	6354737	4895567	7719636
L-lysine	582956	463776	360100	465812	806131	600286	642685	718441	472182	536090	373525	542847
L-methionine	936779	879792	262683	698037	117522	26994	26023	54914	47968	96970	60804	150504
L-phenylalanine	8641149	7749052	4062540	6054651	2773605	2120235	1737321	2202108	2492144	2135230	2837171	2788470
L-proline	6426087	5863670	7431394	8379210	5952690	4920444	4260070	5237115	5353635	4661896	5457078	5704532
L-threonine	1559574	1501894	815657	1305492	584356	449412	331694	451658	462217	485371	411855	556418
L-tryptophan	34179836	29341934	15643304	22682026	9337091	7388415	5831083	7805330	9611108	7783963	9922408	10433795
L-tyrosine	7439467	6601476	3832023	6603968	3172631	2331310	1922002	2386512	1962329	1916696	1941655	2592297
L-valine	16616528	14271747	8159048	12927261	6511198	5184616	4489852	5552623	4698647	4190017	4530799	5648100
AMP	500204	323832	381710	449967	408555	242301	322334	348950	231466	177191	197621	212193
dAMP	149980	169246	152720	212354	56918	37141	10700	57985	67786	50832	68370	75803
Adenosine	179504	240410	97121	94972	3207024	3282300	2897682	3516796	4420556	5011320	4070454	7361396
Adenine	35978	38498	13614	46814	2599445	1237895	2313344	1491431	4923827	7269782	4918908	8213243
GMP	62749	42999	40341	36084	80037	90779	162032	106224	36080	32585	27990	40581
Guanine	3486	2462	3371	0	177472	155628	282785	229775	309869	481151	349861	506801
CMP	117832	122329	104992	103694	67178	30022	48836	40961	9607	24242	15622	34181
Cytidine	12962	0	5826	4531	801355	817768	742715	960921	63980	93245	75397	61620
Cytosine	22512	10588	7285	9143	36307	35106	37254	50098	44303	39610	32203	48347
Allantoate	3986	0	7244	12765	18257	13934	8938	13496	7470	7729	2541	10249
Pyridoxal	69509	67149	82409	79194	106512	90364	93823	100155	43983	59773	38220	41981
Pyridoxamine 5'-phosphate	52349	64231	31502	27450	22138	7749	15705	18496	23525	15650	21112	18641
Nicotinamide	365491	308368	269760	313971	445085	461019	401449	401892	225784	303336	248515	354877
NAD+	471791	544049	508421	614358	519236	340553	266550	386206	379532	393236	291626	339304
D-Rhamnose	16680840	25219018	27769254	17497266	15703388	26357600	18906758	21236528	15700227	19101534	18837576	24079434
D-Glucono-1-5-lactone 6-phosphate	17620	15572	33091	55542	1247083	1073641	677757	996893	33476	62130	21118	48552
Glutathione disulfide	88578	62671	101804	61881	49051	44382	40257	65549	91660	128237	130900	93027
Cys-Gly	259124	242762	246977	322035	77250	60091	48831	62636	59281	78414	91058	95726
gamma-L-Glutamyl-L-cysteine	74608	51273	47120	39402	18489	5472	16104	6735	22506	41136	34064	47416
Folate	2606	4278	0	0	47069	50374	41640	51406	11796	9441	13946	10043
L-Citrulline	7990	9388	10526	17539	51024	77396	66810	60151	18540	11225	6305	10800
Cadaverine	58461	40945	15485	53362	5986	0	2195	6915	2014	0	0	0
Putrescine	173887	151309	158047	138998	177630	164317	144173	136013	210107	193904	225710	215898
Spermidine	1089757	770008	578086	659091	489336	705900	429592	479359	2017155	1022277	2853389	1352301
Spermine	1222497	1123532	1193104	2063535	1497297	2063300	753993	841021	1617900	2551021	2487966	1438571
Homocarnosine	80013	71681	61077	62593	85490	94179	73755	90255	78966	71594	71530	69237
Creatine	30444446	28342112	32876776	34654436	11466475	9274064	7658478	10010311	16259255	14251531	15225801	16553985
Creatinine	135032	130569	116624	137325	61067	54019	31200	35580	44466	30752	57571	47419
N-Acetylornithine	82926	105883	53546	87262	75874	45122	56596	70397	87897	81474	73225	86150
Taurine	102445	98146	176424	209882	71792	72123	45436	57085	40916	36758	42215	60605
L-Methionine S-oxide	1353473	1294312	841788	1218954	651018	438240	364515	484754	440285	411780	383958	561842
3-Methyleneoxindole	282525	226850	111795	179657	77147	53130	47889	47175	68829	66531	84459	88847
Indole-3-acetaldehyde	40625	40664	21744	24317	14110	7082	6241	11790	9376	8437	17407	21402
Indole-3-acetate	151368	161946	160373	155838	185336	192680	149006	243072	30407	26977	40175	55650
Indolepyruvate	172686	124115	105182	131440	44167	47360	35073	26778	39737	32013	38895	35567
g-Oxalo-crotonate	4690	2166	2028	0	1446580	1571199	1540488	1811832	178236	190960	128153	308675
2-Aminomuconate	214579	199964	222997	313711	38300	27043	21227	33144	40533	64678	28482	55602
Sphingosine	8404317	7650043	3291069	5672290	2533072	1797035	2203390	2034944	2778907	3057142	2276300	2942783
Acetylcholine	540257	503567	805064	903379	265767	229136	175666	224340	106879	96659	104976	137168
Choline	29814186	23574576	20667490	19311790	32413914	24633776	22201254	22837704	20538598	21361154	16491917	24445570
L-Carnitine	5483687	5209009	8477085	10655361	3169660	2911833	2403490	2373763	894349	909492	1010134	1047069
acetyl-carnitine (acyl-C2)	10741133	8820753	10800963	11225421	1776042	2064592	1646783	1901633	827221	769515	966873	879709
propionyl-carnitine (acyl-C3)	1492554	1367265	3776090	3416667	553828	504219	421475	548174	155358	175380	186791	234551
butanoyl-l-carnitine (acyl-C4)	970528	889383	655848	995943	42670	36387	27638	34092	97813	57969	97795	80223
O-tetradecanoyl-L-carnitine (acyl-C14)	1319845	1269626	901796	1087468	65452	68192	37119	59716	60006	59937	89692	91904
L-Palmitoylcarnitine (acyl-C16)	368964	286320	258852	295363	31388	28642	25232	25336	48609	32285	34529	55789
Hexadecenoyl-carnitine (acyl-C16:1)	254481	254905	221121	207753	66944	94661	49141	58906	44565	33030	34445	56621
O-octadecenoyl-L-carnitine (acyl-C18:1)	83157	59955	71968	71980	19707	19239	13046	17220	18525	13112	19981	19177
S-Adenosyl-L-methionine	95320	98700	81863	149918	58293	31908	51495	76496	7897	28269	7902	30053
5-Methylthioadenosine	1154343	976901	561470	745014	338005	300296	299699	346269	446200	345353	398574	621518
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	m/z	r.t. (min)
L-alanine	C00041	90.0551	0.6670
L-arginine	C00062	175.1189	0.6330
L-asparagine	C00152	133.0602	0.6590
L-aspartate	C00049	134.0449	0.6870
L-cysteine	C00097	122.0272	0.7040
L-glutamate	C00025	148.0605	0.6960
L-glutamine	C00064	147.0764	0.6590
L-leucine/isoleucine	C00123	132.1020	0.7610
L-lysine	C00047	147.1128	0.6030
L-methionine	C00073	150.0583	0.7120
L-phenylalanine	C00079	166.0856	1.4020
L-proline	C00148	116.0707	0.6870
L-threonine	C00188	120.0657	0.6640
L-tryptophan	C00078	205.0972	1.7740
L-tyrosine	C00082	182.0810	0.7230
L-valine	C00183	118.0864	0.7100
AMP	C00020	348.070312	0.833
dAMP	C00360	332.077576	0.693
Adenosine	C00212	268.104034	0.709
Adenine	C00147	136.061813	0.758
GMP	C00144	364.065338	0.695
Guanine	C00242	152.056732	0.744
CMP	C00055	324.059174	0.68
Cytidine	C00475	244.092896	1.883
Cytosine	C00380	112.050751	0.689
Allantoate	C00499	177.061554	0.625
Pyridoxal	C00250	168.065201	0.681
Pyridoxamine 5'-phosphate	C00647	249.061249	1.776
Nicotinamide	C00153	123.055412	0.752
NAD+	C00003	664.116943	0.684
D-Rhamnose	C01684	165.076004	2.044
D-Glucono-1-5-lactone 6-phosphate	C01236	259.021362	0.755
Glutathione disulfide	C00127	613.15979	0.695
Cys-Gly	C01419	179.048584	0.703
gamma-L-Glutamyl-L-cysteine	C00669	251.069794	0.696
Folate	C00504	442.147339	1.769
L-Citrulline	C00327	176.102997	0.668
Cadaverine	C01672	103.123146	0.626
Putrescine	C00134	89.107437	0.645
Spermidine	C00315	146.165207	0.662
Spermine	C00750	203.223053	0.628
Homocarnosine	C00884	241.131165	2.574
Creatine	C00300	132.076874	0.677
Creatinine	C00791	114.066322	0.674
N-Acetylornithine	C00437	175.107681	0.683
Taurine	C00245	126.022018	0.713
L-Methionine S-oxide	C02989	166.053177	0.698
3-Methyleneoxindole	C02796	146.060059	1.782
Indole-3-acetaldehyde	C00637	160.075684	1.781
Indole-3-acetate	C00954	176.070587	1.973
Indolepyruvate	C00331	204.063126	0.693
g-Oxalo-crotonate	C03453	159.028061	0.756
2-Aminomuconate	C02220	158.044815	0.744
Sphingosine	C00319	300.289673	2.103
Acetylcholine	C01996	146.117615	0.692
Choline	C00114	104.107155	0.665
L-Carnitine	C00318	162.112503	0.667
acetyl-carnitine (acyl-C2)	C02571	204.123032	0.702
propionyl-carnitine (acyl-C3)	C03017	218.138687	0.74
butanoyl-l-carnitine (acyl-C4)	C02862	232.154312	1.734
O-tetradecanoyl-L-carnitine (acyl-C14)	HMDB05066	372.311005	2.141
L-Palmitoylcarnitine (acyl-C16)	C02990	400.342133	2.469
Hexadecenoyl-carnitine (acyl-C16:1)	HMDB06317	398.327179	2.184
O-octadecenoyl-L-carnitine (acyl-C18:1)	HMDB06351	426.358093	2.546
S-Adenosyl-L-methionine	C00019	399.144409	0.667
5-Methylthioadenosine	C00170	298.096802	1.762
METABOLITES_END
#END