SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma

Abstract

Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Graphical abstract

Figure 1.

Essential genes in BL and ABC-DLBCL. Icons indicate essential genes from CRISPR screens colored by the average CSS in BL (red) or ABC-DLBCL (blue) cell lines. BL cell lines: BL60, RAJI, Ramos, ABC-DLBCL cell lines: HBL-1, TMD8, U2932, HLY1.

Figure 2.

SHMT2 enzymatic activity is essential for BL cell growth and viability. (A) Immunohistochemical staining of SHMT2 in lymphoma biopsies and lymph nodes from healthy individuals. Magnification is indicated with 20× and 63×, respectively. Scale bar for 100 µM is indicated. (B) Quantification of IHC with classification into negative, moderate, or strong expression. GC, germinal center; LN, lymph node; MZ, marginal zone. (C) Competitive growth assay of BL cell lines expressing a constitutive shRNA vector targeting SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) together with RFP in coculture with wild-type cells. Percentages of RFP-positive cells were measured at indicated times, and obtained values were normalized to the values of control shRNA expressing cells (mean ± standard error of the mean [SEM]; n = 3; P < .001 compared with nontargeting control shRNA transduced cells [two-way ANOVA]). (D) Cell cycle analyses of the BL cell lines BL60 and Ramos constitutively expressing an shRNA against SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) (mean ± SEM is shown; n = 4 to 5; ***P < .001 in Student t test). (E) Western blot analyses of cleaved PARP (cPARP), total PARP, and cleaved and total Caspase-3 in BL60 and Ramos cells constitutively expressing an shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtr). GAPDH or β-actin served as loading controls (n = 3-4). For quantification see supplemental Figure 2F. (F) Rescue experiment in which BL60 cells carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl) with GFP as fluorescent reporter were transduced with a lentiviral vector coding for the fluorescent reporter BFP only (empty vector) or BFP and an shRNA-resistant version of SHMT2 (SHMT2res) or a catalytically inactive mutant thereof (SHMT2res-K280A). Percentages of GFP/BFP positive cells were determined over time by flow cytometry and normalized to the values of day 2 after doxycycline induction of shRNA expression. The diagram displays the values obtained at day 6 after induction of knockdown (mean ± SEM; n = 3). ***P < .001 in Student t test. Western blot validation for successful SHMT2 reconstitution is shown below. GAPDH served as loading control (n = 3; for empty vector/shCtrl vs shSHMT2 P < .001 in paired Student t test; for SHMT2res and SHMT2res-K280A, P = ns in paired Student t test; P = .51 and P = .37, respectively). (G) Tumor volume in NSG mice that were subcutaneously transplanted with BL60 carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl). Tumor volumes were measured upon intraperitoneal doxycycline induction (once per day) of shRNAs (mean ± SEM; n = 5; P < .01 in two-way ANOVA; **P < .01 in Bonferroni posttest; ***P < .001 in Bonferroni posttest).

Figure 3.

The metabolic effect of SHMT2 inhibition. (A) Metabolites significantly regulated upon inducible SHMT2 knockdown (shSHMT2#2, d3 upon doxycyline induction) from LC/MS analysis (n = 6 each). The heatmap is color coded by row-wise z-scores of metabolite abundances. AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; GSH, glutathione; IMP, inosine monophosphate. (B) Metabolite abundance for selected significantly regulated metabolites from LC/MS analysis (up to n = 6 each; median is shown). (C) Cell viability assay (MTT assay) for BL60 and SU-DHL4 after 48 hours of SHIN1 treatment and supplementation with indicated metabolites. (0.1 mM glycine and 0.3 mM serine correspond to regular medium concentration.) Nucleoside components are described in the supplemental Methods. Data were normalized to medium control (n = 3-4; mean ± SEM is shown). Glycine supplementation: [BL60] P = .0052, [SU-DHL4] ns (two-way ANOVA), Glycine/formate supplementation: [BL60] P = .0032, [SU-DHL4] ns (two-way ANOVA). Bonferroni posttest for glycine supplementation at 3.3 mM: [BL60] 1 µM SHIN1 P < .01; 5 and 10 µM SHIN1 P < .001; [SU-DHL4] 10 µM SHIN1 P < .001; for glycine/formate supplementation at 3.3/2 mM: [BL60] 1 µM SHIN1 P < .01; 5 to 10 µM SHIN1 P < .001; [SU-DHL4] 5 µM SHIN1 P < .05, 10 µM SHIN1 P < .001.

Figure 4.

TCF3 expression depends on SHMT2 function. (A) Integration of CRISPR/Cas9 drop out screen results (CSS, CRISPR screen score) in BL60 cells with total proteome changes (SILAC log fold change [LFC]) in inducible SHMT2 knockdown BL60 cells (shSHMT2#2) on day 5 after induction of shRNA expression. Selected gene names/proteins are indicated. (B) Schematic illustration of the functional role of TCF3 in BL adapted from Schmitz et al. Indicated mutation frequencies were obtained from Schmitz et al, 2012; Love et al, 2012; Grande et al, 2019; Bouska et al, 2017; López et al, 2019. (C) Representative western blot shows inducible TCF3 knockdown in BL60 cell line on day 5 after induction of shRNA expression. GAPDH served as loading control (n = 3; Student t test, shCtrl vs shTCF3.1, P = .003; shCtrl vs shTCF3.2, P = .017; shCtrl vs shTCF3.3, P = .005). (D) Coculture experiment with BL60 TCF3 doxycycline-inducible knockdown cells (3 different shRNA constructs) compared with a nontargeting control shRNA (shCtrl). Cells were cocultured with wild-type cells and analyzed by flow cytometry using the GFP reporter of the vector. GFP-positive cells were normalized to day 1 upon induction (n = 3, mean ± SEM; P < .001 in two-way ANOVA). (E) Cell cycle analysis of the BL60 cell line expressing an inducible shRNA against TCF3 (shTCF3#3) or a nontargeting control shRNA (shCtrl; n = 3, mean ± SEM; **P < .01 in Student t test, ***P < .001 in Student t test). (F) Representative western blots of lysates derived from BL60 and Ramos cells at day 8 upon SHMT2 knockdown (n = 3-4; TCF3 [BL60] P = .02 [Ramos] P = .002, SHP-1 [BL60] P = .02 [Ramos] ns). GAPDH served as loading control. (G) Western blots show SYK Tyr525/526 and AKT Ser473 phosphorylation levels upon inducible knockdown (shTCF3#3) in BL60 cell line 48 hours after induction of shRNA expression compared with a nontarget control shRNA (shCtrl). GAPDH served as loading control. Phosphorylation levels were normalized to SYK or AKT expression, respectively (n = 3; P < .04 in Student t test for pSYK). (H) TCF3 reconstitution using a retroviral vector with GFP fluorescent reporter in constitutive SHMT2 knockdown cells (shSHMT2#1) with an RFP fluorescent reporter in BL60 cell line. Double-positive (GFP/RFP) cells were followed up in flow cytometry (n = 4, mean ± SEM; *P < .05 in Student t test). (I) Phosphoproteomic SILAC-based LC-MS analysis in inducible SHMT2 and CD79a knockdown vs control BL60 cells upon doxycycline induction. SILAC ratios are average of 2 replicates. Differentially phosphorylated sites were defined as phosphosites quantified in at least 2 replicates exhibiting an absolute log2 SILAC ratio >0.5. The Pearson’s correlation coefficient is shown. P value is from a Pearson’s correlation test. (J) SILAC log2 fold change (LFC) for BCR effector protein expression and tyrosine phosphorylation of the indicated BCR effectors upon inducible SHMT2 knockdown vs control in BL60 cells on day 5 after induction of shRNA expression. Differentially phosphorylated phosphosites (Benjamini-Hochberg adjusted P value < 1e-3) are colored in red. SILAC ratios are average of 3 and 2 biological replicates for total proteome and phosphoproteome, respectively. (K) Cell viability (XTT assay) of BL60 cells expressing either a constitutively active variant of the catalytic PI3K subunit P110α (MP110*) or the empty vector (control) that were treated with SHIN1 at a final concentration of 2 µM or dimethyl sulfoxide (DMSO) at day 0. Background corrected absorbance is shown (n = 4, mean ± SEM; on day 4 control + DMSO vs control + SHIN1, P < .001; control + DMSO vs MP110* + DMSO, P < .01; MP110* + DMSO vs MP110*+ SHIN1 ns in Bonferroni posttest).

Figure 4.

TCF3 expression depends on SHMT2 function. (A) Integration of CRISPR/Cas9 drop out screen results (CSS, CRISPR screen score) in BL60 cells with total proteome changes (SILAC log fold change [LFC]) in inducible SHMT2 knockdown BL60 cells (shSHMT2#2) on day 5 after induction of shRNA expression. Selected gene names/proteins are indicated. (B) Schematic illustration of the functional role of TCF3 in BL adapted from Schmitz et al. Indicated mutation frequencies were obtained from Schmitz et al, 2012; Love et al, 2012; Grande et al, 2019; Bouska et al, 2017; López et al, 2019. (C) Representative western blot shows inducible TCF3 knockdown in BL60 cell line on day 5 after induction of shRNA expression. GAPDH served as loading control (n = 3; Student t test, shCtrl vs shTCF3.1, P = .003; shCtrl vs shTCF3.2, P = .017; shCtrl vs shTCF3.3, P = .005). (D) Coculture experiment with BL60 TCF3 doxycycline-inducible knockdown cells (3 different shRNA constructs) compared with a nontargeting control shRNA (shCtrl). Cells were cocultured with wild-type cells and analyzed by flow cytometry using the GFP reporter of the vector. GFP-positive cells were normalized to day 1 upon induction (n = 3, mean ± SEM; P < .001 in two-way ANOVA). (E) Cell cycle analysis of the BL60 cell line expressing an inducible shRNA against TCF3 (shTCF3#3) or a nontargeting control shRNA (shCtrl; n = 3, mean ± SEM; **P < .01 in Student t test, ***P < .001 in Student t test). (F) Representative western blots of lysates derived from BL60 and Ramos cells at day 8 upon SHMT2 knockdown (n = 3-4; TCF3 [BL60] P = .02 [Ramos] P = .002, SHP-1 [BL60] P = .02 [Ramos] ns). GAPDH served as loading control. (G) Western blots show SYK Tyr525/526 and AKT Ser473 phosphorylation levels upon inducible knockdown (shTCF3#3) in BL60 cell line 48 hours after induction of shRNA expression compared with a nontarget control shRNA (shCtrl). GAPDH served as loading control. Phosphorylation levels were normalized to SYK or AKT expression, respectively (n = 3; P < .04 in Student t test for pSYK). (H) TCF3 reconstitution using a retroviral vector with GFP fluorescent reporter in constitutive SHMT2 knockdown cells (shSHMT2#1) with an RFP fluorescent reporter in BL60 cell line. Double-positive (GFP/RFP) cells were followed up in flow cytometry (n = 4, mean ± SEM; *P < .05 in Student t test). (I) Phosphoproteomic SILAC-based LC-MS analysis in inducible SHMT2 and CD79a knockdown vs control BL60 cells upon doxycycline induction. SILAC ratios are average of 2 replicates. Differentially phosphorylated sites were defined as phosphosites quantified in at least 2 replicates exhibiting an absolute log2 SILAC ratio >0.5. The Pearson’s correlation coefficient is shown. P value is from a Pearson’s correlation test. (J) SILAC log2 fold change (LFC) for BCR effector protein expression and tyrosine phosphorylation of the indicated BCR effectors upon inducible SHMT2 knockdown vs control in BL60 cells on day 5 after induction of shRNA expression. Differentially phosphorylated phosphosites (Benjamini-Hochberg adjusted P value < 1e-3) are colored in red. SILAC ratios are average of 3 and 2 biological replicates for total proteome and phosphoproteome, respectively. (K) Cell viability (XTT assay) of BL60 cells expressing either a constitutively active variant of the catalytic PI3K subunit P110α (MP110*) or the empty vector (control) that were treated with SHIN1 at a final concentration of 2 µM or dimethyl sulfoxide (DMSO) at day 0. Background corrected absorbance is shown (n = 4, mean ± SEM; on day 4 control + DMSO vs control + SHIN1, P < .001; control + DMSO vs MP110* + DMSO, P < .01; MP110* + DMSO vs MP110*+ SHIN1 ns in Bonferroni posttest).

Figure 5.

SHMT2 inhibition induces autophagic degradation of TCF3. (A) Representative western blot analysis showing LC3 levels in BL60 cells expressing either SHMT2-specific shRNA or nonspecific control shRNA that were treated with chloroquine at a concentration of 100 µM for 4 hours as indicated. GAPDH served as loading control. (n = 4; P = .01 in Student t test). (B) GFP/RFP ratio of BL60 cells transduced with tfLC3 reporter upon treatment with SHIN1 at a concentration of 2.5 µM, AZD2014 at a concentration of 200 nM, and Torin 1 (TOR1) at a concentration of 500 nM for indicated durations. A reduced ratio represents an increased level of autophagy. Bafilomycin A1 (Baf) treatment at 50 nM was used to inhibit autophagy (n = 3, mean ± SEM; P < .0001 in Tukey’s multiple comparison test for TOR1, AZD2014, and SHIN1 compared with DMSO after 6 hours and 24 hours; P = ns for rescue with Baf after 6 hours and 24 hours). (C) Representative confocal images of LC3 immunofluorescence staining. BL60 cells were treated for 24 hours with 2.5 µM SHIN1 or DMSO as well as for the last 6 hours with 50 nM of Bafilomycin or DMSO and stained for LC3 and DAPI. LC3 was stained with Alexa Fluor 647 (red) and nuclei were counterstained with DAPI (blue). Representative images display the overlay max intensity of the 41 z-stacks of the 647 channel and the average intensity of the z-stacks of the DAPI signal. (D) Quantification of LC3 punctae from microscopic images of BL60 cells treated as described in Figure 5C. Data were normalized to control and reported as percentage (n = 2, with n ≥ 31 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots. P < .0001 according to a Kruskal-Wallis test. (E) Representative western blot analysis showing LC3 levels in BL60 cells upon SHIN1 treatment at 2.5 µM for 48 hours in regular medium and upon supplementation with formate and glycine. Chloroquine treatment was applied at a concentration of 100 µM for 4 hours. GAPDH served as loading control (n = 3; P = .03 in Student t test for ΔLC3-II in SHIN1 vs DMSO in regular medium). (F-G) Representative western blots in BL60 cells showing ULK1 Ser757 phosphorylation, ULK1, TCF3, and SHP-1 after treatment of BL60 cells with 2.5 µM SHIN1 for 48 hours in regular medium and upon supplementation with glycine and formate (3.3 mM and 2 mM, respectively). pULK1 and ULK1 were probed on different membranes, but samples were derived from the same experiment and blots were processed in parallel. GAPDH served as loading control (n = 3; P = .02 in Student t test for pULK1 levels in SHIN1 vs DMSO in regular medium; P = .003 in Student t test for TCF3 levels in regular medium vs glycine/formate supplementation; and P = .008 in Student t test for SHP-1 levels in regular medium vs glycine/formate supplementation). (H) Representative western blots showing TCF3 levels in ATG5 KO compared with control-sgRNA in BL60 cell line upon induction of knockout with 250 ng/mL of doxycycline and 48 hours of SHIN1 treatment at a concentration of 2.5 µM vs DMSO control. GAPDH served as loading control (n = 3; P = .037 in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgCtrl; P = ns in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgATG5). (I) PLA score is shown for PLA of TCF3 and LC3 in SHIN1-treated BL60 cells at a concentration of 2.5 µM for 18 hours compared with DMSO control, in regular medium as well as upon supplementation of glycine/formate at a concentration of 3.3 mM and 2 mM, respectively (n = 4; n ≥ 105 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots (P < .001 in Kruskal-Wallis test). (J) Representative confocal images from PLA for TCF3 and LC3 in BL60 cell line, as described in Figure 5I. Merged images represent the composite images of the PLA of TCF3 and LC3 (red) and the DAPI signal (blue).

Figure 5.

SHMT2 inhibition induces autophagic degradation of TCF3. (A) Representative western blot analysis showing LC3 levels in BL60 cells expressing either SHMT2-specific shRNA or nonspecific control shRNA that were treated with chloroquine at a concentration of 100 µM for 4 hours as indicated. GAPDH served as loading control. (n = 4; P = .01 in Student t test). (B) GFP/RFP ratio of BL60 cells transduced with tfLC3 reporter upon treatment with SHIN1 at a concentration of 2.5 µM, AZD2014 at a concentration of 200 nM, and Torin 1 (TOR1) at a concentration of 500 nM for indicated durations. A reduced ratio represents an increased level of autophagy. Bafilomycin A1 (Baf) treatment at 50 nM was used to inhibit autophagy (n = 3, mean ± SEM; P < .0001 in Tukey’s multiple comparison test for TOR1, AZD2014, and SHIN1 compared with DMSO after 6 hours and 24 hours; P = ns for rescue with Baf after 6 hours and 24 hours). (C) Representative confocal images of LC3 immunofluorescence staining. BL60 cells were treated for 24 hours with 2.5 µM SHIN1 or DMSO as well as for the last 6 hours with 50 nM of Bafilomycin or DMSO and stained for LC3 and DAPI. LC3 was stained with Alexa Fluor 647 (red) and nuclei were counterstained with DAPI (blue). Representative images display the overlay max intensity of the 41 z-stacks of the 647 channel and the average intensity of the z-stacks of the DAPI signal. (D) Quantification of LC3 punctae from microscopic images of BL60 cells treated as described in Figure 5C. Data were normalized to control and reported as percentage (n = 2, with n ≥ 31 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots. P < .0001 according to a Kruskal-Wallis test. (E) Representative western blot analysis showing LC3 levels in BL60 cells upon SHIN1 treatment at 2.5 µM for 48 hours in regular medium and upon supplementation with formate and glycine. Chloroquine treatment was applied at a concentration of 100 µM for 4 hours. GAPDH served as loading control (n = 3; P = .03 in Student t test for ΔLC3-II in SHIN1 vs DMSO in regular medium). (F-G) Representative western blots in BL60 cells showing ULK1 Ser757 phosphorylation, ULK1, TCF3, and SHP-1 after treatment of BL60 cells with 2.5 µM SHIN1 for 48 hours in regular medium and upon supplementation with glycine and formate (3.3 mM and 2 mM, respectively). pULK1 and ULK1 were probed on different membranes, but samples were derived from the same experiment and blots were processed in parallel. GAPDH served as loading control (n = 3; P = .02 in Student t test for pULK1 levels in SHIN1 vs DMSO in regular medium; P = .003 in Student t test for TCF3 levels in regular medium vs glycine/formate supplementation; and P = .008 in Student t test for SHP-1 levels in regular medium vs glycine/formate supplementation). (H) Representative western blots showing TCF3 levels in ATG5 KO compared with control-sgRNA in BL60 cell line upon induction of knockout with 250 ng/mL of doxycycline and 48 hours of SHIN1 treatment at a concentration of 2.5 µM vs DMSO control. GAPDH served as loading control (n = 3; P = .037 in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgCtrl; P = ns in paired Student t test for TCF3 levels in SHIN1 vs DMSO in samples with sgATG5). (I) PLA score is shown for PLA of TCF3 and LC3 in SHIN1-treated BL60 cells at a concentration of 2.5 µM for 18 hours compared with DMSO control, in regular medium as well as upon supplementation of glycine/formate at a concentration of 3.3 mM and 2 mM, respectively (n = 4; n ≥ 105 single cells per condition). Box plots represent the median and 25th to 75th percentiles, whiskers display 10th to 90th percentiles, and outliers are displayed as dots (P < .001 in Kruskal-Wallis test). (J) Representative confocal images from PLA for TCF3 and LC3 in BL60 cell line, as described in Figure 5I. Merged images represent the composite images of the PLA of TCF3 and LC3 (red) and the DAPI signal (blue).

Figure 6.

Identification of drugs acting synergistically with an SHMT inhibitor. (A) IC₅₀ of SHIN1 in different BL cell lines measured by MTT assay. Cells were treated for 4 days (n = 4; mean ± SEM). The presence/absence of reported oncogenic ID3 and TCF3 mutations for the individual cell lines is indicated by + or −. (B) Stable cell lines isolated from murine Eµ:Myc (M2121, M452) and DMBC (BSQ12, BSQ27) tumors were treated with varying doses of SHIN2 for 72 hours, and viability was measured by CellTiterGlo assay (n = 3). Error bars represent standard deviation (SD). For each concentration, both Eµ:Myc lines were compared with both DMBC lines by Welch t test. *P < .05 for all comparisons at that concentration. (C) Results from “spiked-in,” quantitative high-throughput drug screening utilizing a mechanistically annotated library (MIPE 5.0), either synergizing or antagonizing SHIN1 treatment. (D) Selected hits from “spiked-in,” quantitative high-throughput drug screening that are synergistic with SHIN1 treatment. (E) Combined treatment of SHIN1 and methotrexate (MTX) shows synergy in MTT assay (n = 3; 1 representative analysis is shown). eob, excess over bliss. (F) Western blot of BL60 wild-type cells treated with either SHIN1 (concentrations ranging from 0.5-5 µM) or MTX (10-100 nM) for 3 days. GAPDH served as loading control. Cropped blots of representative experiments are shown with quantification for SHIN1-treated cells (n = 3-5; P = .002 in Student t test for altered TCF3 levels in 2.5 µM concentration and P = .003 for SHP-1 levels, respectively). (G) Annexin V staining in CD19⁺ cells derived from bone marrow of a 27-year-old patient with BL after in vitro treatment with SHIN1 (5 µM) and MTX (20 nM) for 96 hours. Cells were normalized to DMSO control. ID3 mutations (L64F, V55fs) were detected by exome sequencing (supplemental Figure 7A; supplemental Table 4). F, phenylalanine; fs, frame shift; L, leucin; V, valine.

Figure 6.

Identification of drugs acting synergistically with an SHMT inhibitor. (A) IC₅₀ of SHIN1 in different BL cell lines measured by MTT assay. Cells were treated for 4 days (n = 4; mean ± SEM). The presence/absence of reported oncogenic ID3 and TCF3 mutations for the individual cell lines is indicated by + or −. (B) Stable cell lines isolated from murine Eµ:Myc (M2121, M452) and DMBC (BSQ12, BSQ27) tumors were treated with varying doses of SHIN2 for 72 hours, and viability was measured by CellTiterGlo assay (n = 3). Error bars represent standard deviation (SD). For each concentration, both Eµ:Myc lines were compared with both DMBC lines by Welch t test. *P < .05 for all comparisons at that concentration. (C) Results from “spiked-in,” quantitative high-throughput drug screening utilizing a mechanistically annotated library (MIPE 5.0), either synergizing or antagonizing SHIN1 treatment. (D) Selected hits from “spiked-in,” quantitative high-throughput drug screening that are synergistic with SHIN1 treatment. (E) Combined treatment of SHIN1 and methotrexate (MTX) shows synergy in MTT assay (n = 3; 1 representative analysis is shown). eob, excess over bliss. (F) Western blot of BL60 wild-type cells treated with either SHIN1 (concentrations ranging from 0.5-5 µM) or MTX (10-100 nM) for 3 days. GAPDH served as loading control. Cropped blots of representative experiments are shown with quantification for SHIN1-treated cells (n = 3-5; P = .002 in Student t test for altered TCF3 levels in 2.5 µM concentration and P = .003 for SHP-1 levels, respectively). (G) Annexin V staining in CD19⁺ cells derived from bone marrow of a 27-year-old patient with BL after in vitro treatment with SHIN1 (5 µM) and MTX (20 nM) for 96 hours. Cells were normalized to DMSO control. ID3 mutations (L64F, V55fs) were detected by exome sequencing (supplemental Figure 7A; supplemental Table 4). F, phenylalanine; fs, frame shift; L, leucin; V, valine.