Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a Percentage of PHGDH activity in Eµ- Myc (#688) cells treated with DMSO or indicated concentrations of the PHGDH inhibitor BI-4916 for 24 h in Asn-containing medium ( n = 3 independent experiments). b Percentage of dead Eµ- Myc (#688) cells (DAPI+) following 24 h of treatment with DMSO, ASNase (0.003 IU/ml) and/or BI-4916 (10 µM) in Asn-containing medium ( n = 4 independent experiments). c Four-day proliferation of Eµ- Myc (#688) cells treated as in ( b ) ( n = 5 independent experiments). d Total protein extracts prepared from Eµ- Myc (#506) cells stably expressing shRNA targeting the firefly luciferase (shSCR) or the murine Phgdh mRNA (two independent sh Phgdh #1 and #2) were immunoblotted for indicated proteins. e Relative quantification of immunoblots presented in ( d ). ( n = 4 independent experiments). f Percentage of dead Eµ- Myc (#506) cells (DAPI+) silenced (sh Phgdh #1 or #2) or not (shSCR) for Phgdh mRNA following 24 h incubation in Asn-containing medium supplemented (+) or not (−) with ASNase (0.003 IU/ml) ( n = 4 independent experiments). g Four-day proliferation of Eµ- Myc cells silenced or not (shSCR) for Phgdh mRNA, treated as in ( f ) ( n = 5 independent experiments). h Survival curves of WT C57BL/6 mice intravenously injected with Eµ- Myc (#506) cells stably expressing shRNA control (shSCR) or targeting the murine Phgdh mRNA (sh Phgdh #1), treated with Vehicle or ASNase every 48 h from day 7 until disease endpoint ( n = 10 mice/group). i Total protein extracts prepared from Eµ- Myc cells isolated from BCL of C57BL/6 mice presented in h , were immunoblotted for the indicated proteins (shSCR-Vehicle, n = 3; shSCR-ASNase, n = 3; sh Phgdh #1-Vehicle, n = 4; sh Phgdh #1-ASNase, n = 4 mice). V Vehicle, A ASNase. The samples derive from the same experiments but different gels for PHGDH, PSAT1, ERK2, and another for ASNS and ERK2 were processed in parallel. j Relative quantification of PHGDH protein levels presented in ( i ) Data are normalized to the control condition shSCR-Vehicle. Data are expressed as mean ± SD ( a , b , e , f , j ) or ± SEM ( c , g ). P -values are from one-way Anova followed by Tukey’s test ( a , e ), 2-way Anova followed by Tukey’s t -test ( b , f , j ), t -test ( c , g ), log-rank test ( h ), and indicated as ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. For detailed individual P -value, please refer to the table.

Article Snippet: A total of 250.000 live Eμ- Myc cells/ml were treated with either DMSO (0.075%), or E-Coli L-asparaginase (0.003 IU/ml), or indicated concentration of PHGDH inhibitor BI-4916 (HY-126253, MCE), BI-4924 (HY-126254, MCE), NCT-503 (HY-101966, MCE), CBR-5884 (HY-100012, MCE), PKUMDL-WQ2101 (HY-123269, MCE), or Olaparib (AZD2281, HY-10162, MCE), or the combination of indicated drugs, in asparagine-free medium supplemented or not with 0.37 mM of L-asparagine (A0884, Sigma) at 37 °C, 5% CO 2 for the indicated time.

Techniques: Activity Assay, Stable Transfection, Expressing, shRNA, Luciferase, Quantitative Proteomics, Western Blot, Incubation, Injection, Control, Isolation

Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a Relative ROS levels in Eµ- Myc (#688) cells cultivated for 15 h in Asn-free medium with (+) or without (–) Asn, ASNase (0.003 IU/ml) and N-acetyl-L-cysteine (NAC, 10 mM, 1 h) ( n = 4 independent experiments). b GSSG/GSH ratio in Eµ- Myc (#688) cells cultivated in Asn-free medium, with (+) or without (−) Asn for 24 h ( n = 4 biological replicates). c NRF2 and ATF4 expression levels in Eµ- Myc cells cultivated for 24 h as in ( a ). d Relative quantification of ( c ) (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 2 independent experiments). e Relative mRNA expression levels in Eµ- Myc (#688) cells cultivated as in ( c ) for 48 h ( n = 3 independent experiments). f , g . Relative ROS levels in Eµ- Myc (#688) cells treated for 20 h in Asn-free medium with (+) or without (−) Asn (0.37 mM) and/or BI-4916 (10 µM) ( f ) or in Asn-containing medium with (+) or without ((−), DMSO), ASNase (0.003 IU/ml) and/or BI-4916 (10 µM) ( g ) ( n = 4 independent experiments). h Percentage of dead Eµ- Myc (#506) cells (DAPI+) treated with (+) or without (−) DMSO, ASNase (0.003 IU/ml), BI-4916 (7.5 µM) and/or NAC (5 mM) for 48 h in Asn-containing medium ( n = 4 independent experiments). i Heatmap representation of mRNA expression levels for the indicated genes, determined by RNAseq from Eµ- Myc cells isolated from 12 individual Eµ- Myc Tg/+ mice. j As in ( i ) in BCL harvested from Eµ- Myc cells-bearing mice treated with Vehicle or ASNase ( n = 1 /group). k slc7a11 mRNA expression levels (transcript per million) in selected tumor entities available on the TCGA database. l Total protein extracts from Eµ- Myc (#506) cells stably expressing control (CTL) or murine SLC7A11-encoding (SLC7A11 OE) vectors were immunoblotted for the indicated proteins. The samples derive from the same experiments but different gels for SC7A11, ERK2, and another for CD98 were processed in parallel. m Relative quantification of immunoblots presented in ( l ). Data normalized to SLC7A11 OE condition ( n = 3 independent experiments). n Percentage of dead CTL and SLC7A11 OE-overexpressing Eµ- Myc (#506) cells (DAPI+) treated as in ( h ) ( n = 5 independent experiments). Data are expressed as mean ± SD ( a , d – h , m , n ) or ± SEM ( b ). P -values are from 2-way Anova followed by Tukey’s test ( a , f – h , n ), t -test ( b , m ), one-way Anova followed by Tukey’s test ( d , e ), and indicated as ns , not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001 ****, p < 0.0001. For detailed individual P -value, please refer to the table.

Article Snippet: A total of 250.000 live Eμ- Myc cells/ml were treated with either DMSO (0.075%), or E-Coli L-asparaginase (0.003 IU/ml), or indicated concentration of PHGDH inhibitor BI-4916 (HY-126253, MCE), BI-4924 (HY-126254, MCE), NCT-503 (HY-101966, MCE), CBR-5884 (HY-100012, MCE), PKUMDL-WQ2101 (HY-123269, MCE), or Olaparib (AZD2281, HY-10162, MCE), or the combination of indicated drugs, in asparagine-free medium supplemented or not with 0.37 mM of L-asparagine (A0884, Sigma) at 37 °C, 5% CO 2 for the indicated time.

Techniques: Expressing, Quantitative Proteomics, RNA sequencing, Isolation, Stable Transfection, Control, Western Blot

Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a Total protein extracts prepared from Eµ- Myc #688 cells cultured in Asn-free medium, supplemented (+) or not (−) with Asn (0.37 mM) and ASNase (0.003 IU/ml) for 24 h, were immunoblotted for the indicated proteins. Etoposide treatment (1 µg/ml) for 3 h was used as a positive control. b Relative quantification of immunoblots presented in ( a ). (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 3 independent experiments). c As in a for the indicated proteins expressed in Eµ- Myc #506 and #688 cells. From #506 cells, the samples derive from the same experiments, but different gels for P-ATR (S428), P-Chk1 (S345), KAP1, ERK2, another for ATR, another for Chk1, P-RPA32 (S33), P-KAP1 (S824), another for RPA32, and another for γH2AX were processed in parallel. From #688 cells, the sample derived from the same experiments, but different gels for P-ATR (S428), P-Chk1 (S345), P-KAP1 (S824), ERK2, another for ATR, Chk1, KAP1, another for P-RPA32 (S33), another for RPA32, and another for γH2AX were processed in parallel. d As in ( b ) for immunoblots presented in ( c ) (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 3 independent experiments). e γH2AX expression levels in Eµ- Myc cells (#506 and #688) following 24 h incubation in Asn-containing medium supplemented (+) or not (−) with ASNase (0.003 IU/ml) and/or N-acetyl-L-cysteine (NAC, 10 mM). f Relative quantification of γH2AX expression in Eµ- Myc cells presented in ( e ) (#688 cells, triangle, n = 2 independent experiments; #506 cells, square, n = 1). g γH2AX expression levels in Eµ- Myc (#688) cells treated (+) or not (−) for 24 h with DMSO (−/−), ASNase (0.003 IU/ml) and/or BI-4916 (10 µM). h Relative quantification of γH2AX expression levels in Eµ- Myc cells presented in ( g ). (#688 cells, triangle, n = 2 independent experiments; #506 cells, square, n = 1). i Total protein extracts prepared from BCL harvested from C57BL/6 mice engrafted with control (CTL) or V5-tagged murine PHGDH-overexpressing (PHGDH OE) Eµ- Myc (#506) cells and treated with Vehicle or ASNase from day 7 until disease endpoint, were immunoblotted for the indicated proteins (CTL-Vehicle, n = 3; CTL-ASNase, n = 3; PHGDH OE-Vehicle, n = 4; PHGDH OE-ASNase, n = 4 mice). The samples derive from the same experiments, but different gels for γH2AX, ERK2 (upper), another for P-ChK1 (S345), ERK2 (middle), another for Chk1, another for PHGDH, ERK2 (lower), and another for V5 were processed in parallel. j Relative quantification of γH2AX and P-Chk1 (Ser345) expression levels in BCL presented in ( i ). V Vehicle, A ASNase. For all graphs, data are expressed as mean ± SD. P -values are from one-way Anova followed by Tukey’s test ( b , d ), 2-way Anova followed by Tukey’s test ( f , h , j ) and indicated as ns , not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 ****, p < 0.0001. For detailed individual P -value, please refer to the table.

Article Snippet: A total of 250.000 live Eμ- Myc cells/ml were treated with either DMSO (0.075%), or E-Coli L-asparaginase (0.003 IU/ml), or indicated concentration of PHGDH inhibitor BI-4916 (HY-126253, MCE), BI-4924 (HY-126254, MCE), NCT-503 (HY-101966, MCE), CBR-5884 (HY-100012, MCE), PKUMDL-WQ2101 (HY-123269, MCE), or Olaparib (AZD2281, HY-10162, MCE), or the combination of indicated drugs, in asparagine-free medium supplemented or not with 0.37 mM of L-asparagine (A0884, Sigma) at 37 °C, 5% CO 2 for the indicated time.

Techniques: Cell Culture, Positive Control, Quantitative Proteomics, Western Blot, Derivative Assay, Expressing, Incubation, Control