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. 2018 Jul;559(7715):632-636.
doi: 10.1038/s41586-018-0316-7. Epub 2018 Jul 11.

Histidine Catabolism Is a Major Determinant of Methotrexate Sensitivity

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Free PMC article

Histidine Catabolism Is a Major Determinant of Methotrexate Sensitivity

Naama Kanarek et al. Nature. .
Free PMC article

Abstract

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.

Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Loss of FTCD decreases the sensitivity of cancer cells to methotrexate
a. Relative daily cell counts of the mixed culture of 42 genomically-barcoded hematopoietic cancer cell lines. List of cell lines can be found in Source Data_1, sheet “barcoded cell lines”. We focused our efforts on hematopoietic cell lines because methotrexate is most commonly used to treat hematopoietic malignancies. Cells were co-cultured and treated with three concentrations of methotrexate (0.1, 0.5, or 5 μM). Cell counts are presented relative to the vehicle-treated co-culture. n=3, biological replicates. Error bars indicate SD. b. Sequencing results of the competitive co-culture experiment. Heatmap of relative barcode abundance for each indicated cell line following treatment with 5 μM methotrexate or vehicle after 2, 4, or 6 days compared to those in the initial cultures. Raw data of sequencing results can be found in Source Data_1 sheet “Counts_sequencing”. c. Validation of the results of the competitive co-culture experiment as shown by individual dose-response curves of seven relatively sensitive cell lines (MOLM13, EOL-1, SUPM2, DEL, HEL, NALM6, SUDHL1), and six relatively resistant cell lines (HL-60, Ramos, HBL-1, Raji, SUDHL8, Jeko-1). n=2, biological replicates. Each biological replicate included technical triplicate. Error bars indicate SD. d. Top 40 genes that scored in the genome-wide CRISPR/Cas9-based positive selection screen. These genes had the largest differential CRISPR scores between vehicle- and methotrexate-treated samples. The heatmap represents the CRISPR score of two biological replicates of each screen (vehicle- and methotrexate-treated). CRISPR score of all genes can be found in Source Data_1, sheet “CRISPR score”. e. Abundance of each of the individual sgRNAs targeting FTCD in the screen. Read counts for each sgRNA is presented for each of the screen biological replicates of the vehicle-treated and methotrexate-treated samples. See also Source Data_1, sheet “screen rpm” for read counts raw data, including the genes HAL and AMDHD1. f. Fold-change in methotrexate EC90 and doxorubicin EC90 in HEL cells stably expressing shRNA targeting either SLC19A1 or FTCD compared to the average of three non-targeting shRNAs (shGFP, shRFP, and shLacZ). n=2, biological replicates. Each biological replicate included technical triplicate. g. Expression levels of SLC19A1 (left) and FTCD (right) in HEL cells stably expressing non-targeting shRNAs (shLacZ and shGFP) or targeting shRNAs (shSLC19A1 and shFTCD). Expression levels were measured by qPCR and normalized to the average of two control genes, UBC and HPRT. n=3, technical replicates. h. Validation of genetic depletion of FTCD by the CRISPR/Cas9 system in HEL, Ramos and LAMA84 cell lines. Expression of FTCD was measured by qPCR and normalized to the average of two control genes, UBC and HPRT. n=3 (except for HEL cells expressing sgFTCD_2 + mFTCD, then n=2 due to loss of RNA from one sample), biological replicate. Error bars indicate SD. Raw data of survival curves of these lines can be found in Source Data_1, sheet “Survival MTX”.
Extended Data Figure 2
Extended Data Figure 2. FTCD depletion enables cancer cells to maintain THF pools and nucleotide synthesis even when treated with methotrexate (part 1)
a. The histidine degradation pathway as previously described,–. Enzymes are marked in blue. b. Metabolites detected by LC/MS and their corresponding retention times. The retention time for each detected metabolite is listed. The LC column used for the detection of each metabolite is also indicated. c. Greater pool of 5-methyl THF in vehicle-treated HEL cells following FTCD depletion. It is not readily clear why, in vehicle-treated HEL cells, FTCD depletion caused a reduction in THF levels (Fig. 2e). However, 5-methyl THF levels in these cells were increased significantly (Extended Data Fig. 2c and 3c), indicating that there was no depletion in the overall recyclable amounts of THF in these cells. 5-methyl THF levels were measured by LC/MS in vehicle-treated HEL and Ramos cells. 5-methyl THF levels were normalized to aminopterin as an internal standard. p-values were calculated using one-way ANOVA. n=3, biological replicates. d. Chemical structures of the folate entities found in cells and mentioned in Fig. 2c. e-f. Labeling rate by [U-13C] serine is not different between FTCD-depleted cells and control cells. Fractional labeling of glycine (e) and serine (f) is unchanged by FTCD depletion in HEL and Ramos cells in vehicle-and methotrexate-treated cells. Glycine and serine levels were normalized to isotopically-labeled glutamate as an internal standard. p-values were calculated for the unlabeled fraction by one-way ANOVA. n=3, biological replicates. Source data for Fig. 2 and Extended Data Fig. 2 and 3 can be found in the file Source Data_2. Abbreviation: UROC1 – urocanate hydratase 1.
Extended Data Figure 3
Extended Data Figure 3. FTCD depletion enables cancer cells to maintain THF pools and nucleotide synthesis even when treated with methotrexate (part 2)
a-b. Pool sizes of glycine (a) and serine (b) are not significantly different between FTCD-depleted and control cells. Glycine and serine levels were measured in vehicle- or methotrexate-treated HEL and Ramos cells. p-values were calculated by one-way ANOVA. n=3, biological replicates. c, d. Greater cellular pool of 5,10-methenyl THF in methotrexate-treated cells following FTCD depletion. [U-13C] serine labeling of 5,10-methenyl THF showed higher abundance of unlabeled 5,10-methenyl THF in FTCD-depleted cells, compared to control cells. This implies higher availability of 5,10-methenyl THF in these cells at the time of labeling onset, which agrees with the higher levels of both THF and 5,10-methenyl THF in the methotrexate-treated FTCD-depleted cells compared to the methotrexate-treated control cells. c. 5,10-methenyl THF levels were measured by LC/MS in vehicle- and methotrexate-treated HEL and Ramos cells. 5,10-methenyl THF levels were normalized to aminopterin as an internal standard. p-values were calculated by one-way ANOVA. n=3 biological replicates. d. HEL cells were treated with 5 μM methotrexate for 48 hours, and Ramos cells were treated with 20 μM methotrexate for 72 hours to decrease THF levels and nucleotide synthesis in WT cells. The media was then replaced with [U-13C] serine-containing media, and cells were incubated in [U-13C] serine plus methotrexate for additional 24 hours followed by cell harvesting and LC/MS analysis. Higher abundance of unlabeled 5,10-methenyl THF implies higher availability of reduced folate at the time of labeling onset. 5,10-methenyl THF levels were normalized to aminopterine as an internal standard. p-values were calculated for the unlabeled fraction by one-way ANOVA. n=3, biological replicates.
Extended Data Figure 4
Extended Data Figure 4. The histidine degradation pathway affects the sensitivity of cancer cells to methotrexate and HAL expression is associated with treatment response in acute lymphoblastic leukemia (ALL) patients (part 1)
a. Genetic depletion of the enzymes HAL and AMDHD1 decreased sensitivity to methotrexate, but not to a control drug, doxorubicin. Cell viability after treatment with varying concentrations of methotrexate and doxorubicin was used to calculate the EC90s. n=3, biological replicates. Raw data of survival curves can be found in Source Data_3, sheet “Survival MTX”. b. Expression levels of AMDHD1 (left) and FTCD (right) were not significantly different across methotrexate-sensitive hematopoietic cell lines compared to methotrexate-resistant cell lines. The response to methotrexate was determined in a pooled fashion using genomically-barcoded cell lines (Fig. 1a and Extended Data Fig. 1a-c). Expression levels of AMDHD1 and FTCD were measured by qPCR and normalized to the average of control genes (UBC and HPRT). p-values were calculated using the KS test. n=4 for resistant cell lines and n=6 for sensitive cell lines (biologically independent samples). Each qPCR included three technical replicates. c. Fractional labeling of glycine by [U-13C] serine is unchanged by HAL depletion in HEL and Ramos cells in vehicle-and methotrexate-treated cells. d. Uptake of [U-13C] serine is higher in methotrexate-treated control cells but not in methotrexate-treated HAL-deficient cells. e. Glycine levels are not significantly different between HAL-deficient and control cells except for HEL cells treated with methotrexate. f. Serine levels are not significantly different between HAL-deficient and control cells except for HEL cells treated with methotrexate, where HAL-deficient HEL cells have similar levels of serine as vehicle-treated cells. For c-f: Glycine and serine levels were normalized to isotopically-labeled glutamate as an internal standard. p-values were calculated for the unlabeled fraction (c, d) or to total values (e, f) by one-way ANOVA. n=3, biological replicates. Source data for Fig. 3b-d and Extended Data Fig. 4c-f can be found in the file Source Data_3, sheet “metabolite profiling”.
Extended Data Figure 5
Extended Data Figure 5. The histidine degradation pathway affects the sensitivity of cancer cells to methotrexate and HAL expression is associated with treatment response in acute lymphoblastic leukemia (ALL) patients (part 2)
a. Validation of genetic depletion of HAL by the CRISPR/Cas9 system in NCIH1666 and EOL-1 cells. Expression levels of HAL were measured by qPCR and normalized to the average of two control genes, UBC and HPRT. p-values were calculated by two-way ANOVA. n=3 technical replicates. Error bars indicate SD. Raw data for survival assays Fig. 3e, f can be found in Source Data_3, sheets “cell lines survival” and “HAL KO survival”. b-d. Expression levels of SLC19A1 (b), AMDHD1 (c), or FTCD (d) do not predict survival of pediatric ALL patients treated with a regimen that includes methotrexate. Kaplan-Meier curves of overall survival of ALL patients with high (top quantile, colored green) and low (bottom quantile, colored blue) expression of each of the assayed genes. Patient sample size for each group is indicated. p-values were calculated using the log-rank (Mantel-Cox) test. Raw data for ALL patients gene expression can be found in Source Data_3, sheet “ALL patients”.
Extended Data Figure 6
Extended Data Figure 6. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate (part 1)
a. In vivo imaging of luciferase-expressing HEL tumor xenografts. Mice were imaged before (top images) and after (bottom image) five days of methotrexate treatment alone or in combination with histidine supplementation. For panels a,c, HEL cell-derived tumor-bearing mice: vehicle n=5, histidine supplementation n=4, methotrexate n=6, methotrexate + histidine supplementation n=6. Four mice per group are presented. b. In vivo imaging of luciferase-expressing SEM tumor xenografts. Mice were imaged before (top images) and after (bottom image) five days of treatment. All mice are presented. Mice are numbered in color by their experimental group. For panels b,d, SEM cell-derived tumor-bearing mice: vehicle n=6, histidine supplementation n=7, methotrexate n=7, methotrexate + histidine supplementation n=7. c. Additional images of H&E analyses of HEL cell-derived tumor sections from methotrexate-treated and methotrexate plus histidine supplementation-treated mice. d. H&E images of SEM cell-derived tumor sections from all treatment groups. Three mice per group are presented. Source data for Fig. 4 and Extended Data Fig. 6–8 can be found in the file Source Data_4.
Extended Data Figure 7
Extended Data Figure 7. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate (part 2)
a. Higher magnification of H&E images of tumor sections from SEM cell-derived tumor-bearing mice from all groups. b. H&E analyses of kidney sections from HEL cell-derived tumor-bearing mice from all experimental groups.
Extended Data Figure 8
Extended Data Figure 8. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate (part 3)
THF levels decreased following methotrexate treatment and decreased even further when methotrexate was combined with histidine supplementation. Methotrexate levels were not different in tumors from mice treated with methotrexate alone or in combination with histidine supplementation. Nucleotide abundance was significantly lower in tumors from mice treated with methotrexate and histidine supplementation compared to tumors from vehicle-treated mice. p-values were calculated using non parametric one-way ANOVA for all comparisons (KW test), except for methotrexate – p values were calculated by two tailed t test. vehicle n=6, histidine n=6, methotrexate n=6, methotrexate + histidine n=7.
Extended Data Figure 9
Extended Data Figure 9. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate (part 4)
a. H&E analyses of liver sections from HEL cell-derived tumor-bearing mice from all experimental groups. b. Weight loss as percentage of the first experimental day, prior to treatment. p-value was calculated using non parametric t test (Mann-Whitney). c, d. Metabolites of the histidine degradation pathway were increased in HEL tumors from mice treated with histidine supplementation. Histidine (left) and FIGLU (right) levels were measured in tumors by LC/MS and normalized to isotopically-labeled histidine as an internal standard. p-values were calculated using non parametric t test (Mann-Whitney). All metabolites measured in tumors were normalized to an average of 4 amino acids (phenylalanine, leucine, valine and tyrosine) as an internal loading control. For HEL cell-derived tumor-bearing mice: vehicle n=5, histidine supplementation n=4, methotrexate n=5, methotrexate + histidine supplementation n=6. For SEM cell-derived tumor-bearing mice: vehicle n=6, histidine supplementation n=6, methotrexate n=6, methotrexate + histidine supplementation n=7 e, f. Plasma levels of methotrexate, histidine, 5-methyl THF and folate. Methotrexate was detected in the plasma of methotrexate-treated mice only (left chart). No significant difference in histidine levels was detected (second chart to the left). 5-methyl THF levels were significantly lower in the plasma of methotrexate-treated mice (second chart to the right). Folate levels increased in the plasma of methotrexate-treated mice (right chart). Metabolite levels were measured from fresh plasma samples by LC/MS and normalized to isotopically-labeled histidine or to aminopterin as an internal standard. p-values were calculated using non parametric one-way ANOVA. Group sizes are the same as in panels c,d.
Extended Data Figure 10
Extended Data Figure 10. In vivo histidine supplementation sensitizes tumors to methotrexate without enhancement of treatment toxicity (part 1)
a. We evaluated whether methotrexate treatment combined with histidine supplementation might be more toxic than methotrexate alone by setting up a longer treatment regime of 15 days with a recovery period of two weeks. During the experiment we monitored weight loss and observed no difference between mice treated with methotrexate alone and those treated with methotrexate and histidine. NOD-SCID mice were injected with HEL cells subcutaneously on day 1, followed by weight measurement every other day, in vivo imaging of HEL cell-derived tumors (on days 7, 15, 20 and 28), treatment on days 12 to 23 (vehicle, histidine and methotrexate injections every other day), and final termination of the experiment after 40 days, unless an early euthanization was required in accordance with the guidelines for humane experimental end-point of the animal care committee at MIT. The experiment included four experimental groups: vehicle-treated (saline) (n=7), histidine supplementation (n=8), methotrexate-treated (n=8), and histidine supplementation combined with methotrexate treatment (n=8). Serum was collected at days zero and day 23 for metabolite profiling and liver diagnostics. Methotrexate dose used was 25 mg/kg based on the weight measured at day 0. Histidine dose was 18 mg per injection in 400 μl saline. b. Significant reduction in tumor size over time in mice treated with the combination of methotrexate and histidine supplementation. Tumors were imaged in vivo by luciferase expression at the indicated days. Fold changes in tumor sizes over measurements done on day 7 are presented. p-values were calculated by non parametric one-way ANOVA. Group size changed over time due to mice euthanization for humane reasons: on day 15: vehicle-treated (n=5), histidine supplementation (n=7), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). on day 20: vehicle-treated (n=6), histidine supplementation (n=6), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). on day 28: vehicle-treated (n=6), histidine supplementation (n=5), methotrexate-treated (n=7), and histidine supplementation combined with methotrexate treatment (n=8). c. in vivo imaging of luciferase-expressing HEL cell-derived tumors at days 7 (top) and 28 (bottom). Mice are numbered in color by their experimental group. All mice that participated in the experiment are shown, group size is the same as in panel a. d, e. No elevation in the abundance of serum markers indicative of kidney damage in methotrexate plus histidine supplementation-treated mice compared to methotrexate-treated mice. Markers of kidney toxicity (urea and creatinine) were measured by LC/MS in serum samples of the tested mice. Urea and creatinine relative abundance was normalized to isotopically-labeled valine and tryptophan as an internal standard. p-values were calculated using non parametric one-way ANOVA. Group size: vehicle-treated (n=7), histidine supplementation (n=7), methotrexate-treated (n=8), and histidine supplementation combined with methotrexate treatment (n=6). f, g. Some elevation in liver-toxicity markers in mice treated with the combined therapy compared to methotrexate alone. Markers of liver toxicity (ALT and AST) were measured by an external serum diagnostics lab (IDEXX) in serum samples of the tested mice. Measurement units are indicated. p-values were calculated using non parametric one-way ANOVA. Group size: vehicle-treated (n=6), histidine supplementation (n=6), methotrexate-treated (n=6), and histidine supplementation combined with methotrexate treatment (n=5). Source data for Extended Data Fig. 9 can be found in the file Source Data_5.
Extended Data Figure 11
Extended Data Figure 11. In vivo histidine supplementation sensitizes tumors to methotrexate without enhancement of treatment toxicity (part 2)
a, b. Histological analyses indicated that the kidney and liver appeared normal at the end of the two week recovery period. See also Supplementary Figure 3a “toxicity test_extra panels”, top panel. a. H&E analyses of kidney sections from mice from all experimental groups. Tissues were collected at the conclusion of the experiment, after two weeks recovery post treatment. Group sizes are the same as in Extended Data Fig. 9a. b. H&E analyses of liver sections from mice from all experimental groups. Group sizes are the same as in Extended Data Fig. 9a. Tissues were collected at the conclusion of the experiment, after two weeks recovery post treatment. See also Supplementary Figure 3b “toxicity test_extra panels”, bottom panel: No differences in the overall tissue morphology between methotrexate-treated mice and mice treated with methotrexate and histidine supplementation in intestines collected immediately at the end of a 5 day treatment. H&E analyses of intestine sections from mice from all experimental groups of mice bearing SEM tumors xenografts. These samples were collected immediately after the conclusion of a 5 day-therapy regime of daily injections, to allow the detection of acute damage to the intestine following the treatment and before recovery. This data show no difference in immediate damage to the intestine of mice treated with either methotrexate alone or methotrexate in combination with histidine supplementation.
Figure 1
Figure 1. Loss of FTCD decreases the sensitivity of cancer cells to methotrexate
a. Selection of the HEL cell line for the CRISPR/Cas9-based screen. The genomes of 42 hematopoietic cancer cell lines were individually barcoded. The cell lines were pooled together and treated with 0, 0.1, 0.5 and 5 μM methotrexate for 6 days. Genomic barcodes were sequenced to determine the relative representation of each line in the mixed culture at the various methotrexate concentrations. The erythroleukemia HEL cell line was identified as a sensitive cell line suitable for a genome-wide, positive-selection CRISPR/Cas9-based screen. b. The two top hits in the CRISPR/Cas9-based screen, were SLC19A1 and FTCD. Genes were ranked by the difference between their CRISPR score in the methotrexate-treated and the vehicle-treated samples. c-e. Targeting FTCD by CRISPR/Cas9 in HEL cells decreased their sensitivity to methotrexate c. Fold change in the methotrexate EC90s of HEL cells treated with methotrexate for 5 days and stably expressing the indicated constructs. Methotrexate EC90s are relative to wild-type (WT) cells (n=3, except for SLC19A1 where n=2, biological replicates). d. HEL cells stably expressing the indicated constructs were counted daily to assess their survival following treatment with 5 μM methotrexate (n=3, biological replicates). e. DIC images of HEL cells stably expressing the indicated constructs and treated with 5 μM methotrexate for three days. Scale bar = 100 μm. Presented is a representative experiment (n=3). f. Loss of FTCD decreased the sensitivity of additional cell lines (Ramos and LAMA84) to methotrexate. Shown are fold changes in the EC90s of methotrexate and the control drug, doxorubicin, compared to WT cells (n=3, biological replicates, ordinary one-way ANOVA, comparing sgFTCD to each of the other samples. For doxorubicin all p values were non-significant). Abbreviations: sgAAVS – cells stably expressing an sgRNA targeting the non-coding AAVS locus,. sgFTCD – cells stably expressing an sgRNA targeting FTCD. mFTCD – cells stably expressing mouse Ftcd cDNA that is not targeted by the sgRNA used in this experiment. sgSLC19A1 – cells stably expressing an sgRNA targeting SLC19A1.
Figure 2
Figure 2. FTCD depletion enables cancer cells to maintain THF pools and nucleotide synthesis even when treated with methotrexate
a. The histidine degradation pathway. FTCD has two functions in the histidine degradation pathway. The FT domain metabolizes THF and the histidine breakdown product FIGLU to produce glutamate and 5-formimino THF, and the CD domain metabolizes 5-formimino THF to 5,10-methenyl THF. b. CRISPR/Cas9-induced FTCD depletion increased levels of histidine (top), decreased levels of 5-formyl THF (bottom) in HEL and Ramos cells, and decreased levels of 5,10-methenyl THF in HEL cells and slightly decreased levels of 5,10-methenyl THF in Ramos cells (middle). c. Utilization of THF by the purine synthesis pathway (left cycle), the TMP synthesis pathway (right external cycle), the methyl cycle (right internal cycle), and by FTCD. 5,10-methenyl THF is metabolized to 5-formyl THF by the enzyme SHMT. d. Greater cellular pool of THF and significantly higher abundance of nucleotides in methotrexate-treated cells following FTCD depletion. THF (top), IMP (middle) and TTP (bottom) levels were measured by LC/MS in vehicle- and methotrexate-treated HEL and Ramos cells. e. Newly-synthesized IMP (top) and TTP (bottom) in FTCD-depleted, methotrexate-treated cells shown by [U-13C] serine labeling. IMP and TTP fractional labeling is shown for vehicle-treated and methotrexate-treated HEL and Ramos cells. Right – the four carbons that are contributed to IMP or the one carbon contributed to TTP by [U-13C] serine. b, d, e - n=3, biological replicates. b, d - one-way ANOVA. e - two-way ANOVA for the unlabeled fractions. Abbreviations: ns – non significant. SHMT – serine hydroxymethyltransferase. TYMS – thymidylate synthase. DHFR – dihydrofolate reductase. MTHFR – Methylene tetrahydrofolate reductase. MS – Methionine synthase. GART – GAR formyltransferase. AICART – AICAR formyltransferase. IMP – inosine monophosphate. TTP – thymidine triphosphate.
Figure 3
Figure 3. The histidine degradation pathway affects the sensitivity of cancer cells to methotrexate and HAL expression is associated with treatment response in acute lymphoblastic leukemia (ALL) patients
a. CRISPR/Cas9-induced HAL depletion decreased the sensitivity of HEL and Ramos cells to methotrexate. Shown is the average fold change in the methotrexate EC90 relative to WT (n=2, biological replicates, one-way ANOVA, as multiple comparisons between sgHAL and each of the other samples). b. Greater cellular pool of THF in methotrexate-treated cells following HAL depletion. Top - THF levels were measured by LC/MS in vehicle- and methotrexate-treated HEL and Ramos cells. Newly synthesized adenosine monophosphate (AMP, middle) and TTP (bottom) in HAL-depleted and control cells, treated with methotrexate, shown by [U-13C] serine labeling (n=3, biological replicates, for THF - one-way ANOVA, for AMP and TTP two-way ANOVA for the unlabeled fraction) c. HAL expression is significantly higher in cells that are more sensitive to methotrexate. The response to methotrexate was determined in a pooled fashion using genomically barcoded cell lines (Fig. 1a). HAL expression was measured by qPCR (n=4 for resistant cell lines and n=6 for sensitive cell lines, biologically independent samples, KS test). d-e. Differences in HAL expression are associated with methotrexate sensitivity in cancer cell lines. d. Cell lines were ranked by HAL expression according to RNAseq data. Green - cell lines with high HAL expression. Blue - cell lines with low HAL expression. e. HAL expression (by qPCR) and methotrexate EC90 are shown for each of the colored cell lines from Fig. 3d (Low HAL - n=3, high HAL - n=7, KS test, individual EC90 values: n=2, biological replicates). f. CRISPR/Cas9- depletion of HAL in EOL-1 and NCIH1666 cells decreased their sensitivity to methotrexate. Shown are fold changes in methotrexate EC90 compare to WT (n=2, biological replicate, one-way ANOVA). g. HAL expression predicts better survival in pediatric ALL patients treated with a regimen that included methotrexate. Kaplan-Meier curves of overall survival of ALL patients with high (top quantile, green) or low (bottom quantile, blue) expression of HAL (n=21).
Figure 4
Figure 4. In vivo histidine supplementation increases flux through the histidine degradation pathway and sensitizes tumors to methotrexate
a. Workflow for the in vivo assessment of the methotrexate-sensitivity of tumor xenografts following histidine supplementation. HEL or SEM cells were injected subcutaneously into NOD-SCID mice. Three weeks later tumor size was assessed by in vivo imaging and mice were randomly divided into four experimental groups: vehicle-treated, histidine supplementation, methotrexate-treated, and histidine supplementation combined with methotrexate treatment. The treatments were followed by a second imaging session. b, c. The combination of methotrexate and histidine supplementation is the only treatment that resulted in a significant reduction in tumor size. Fold changes in tumor size as measured in vivo before and after the different treatments are presented for HEL cell-derived tumors (b), and SEM cell-derived tumors (c) (non-parametric one-way ANOVA, KW test). d. The combination of methotrexate treatment and histidine supplementation caused cancer cell death. Hematoxylin-eosin (H&E) staining of tumors from mice injected with HEL cells and treated with the different regimes is presented at two magnifications. Mitotic cells (blue arrows) and apoptotic or necrotic cells (orange arrows) are marked in the bottom panel. e, f. The combination of methotrexate and histidine supplementation was the only treatment that resulted in significant necrosis in tumors. Necrotic areas as detected by H&E staining (d, top three rows, right column) were measured in the different groups (non parametric one-way ANOVA, KW test). g. THF levels decreased following methotrexate treatment and decreased even further when methotrexate was combined with histidine supplementation. Methotrexate levels were not different in tumors from mice treated with methotrexate alone or in combination with histidine supplementation. Nucleotide abundance was significantly lower in tumors from mice treated with methotrexate and histidine supplementation compared to tumors from vehicle-treated mice (non parametric one-way ANOVA, KW test. Methotrexate –two tailed t test). b, d, e - vehicle n=5, histidine n=4, methotrexate n=6, methotrexate + histidine n=6. c, f - n=6, n=7, n=7, n=7 for the same groups. g - n=5, n=4, n=5, n=6, for the same groups. Abbreviations: AMP – adenosine monophosphate. GMP – guanosine monophosphate. TMP – thymidine monophosphate.

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