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. 2022 Dec 2;21(12):1862-1874.
doi: 10.1158/1535-7163.MCT-22-0004.

NOTCH Signaling Limits the Response of Low-Grade Serous Ovarian Cancers to MEK Inhibition

Marta Llaurado Fernandez #  1 , E Marielle Hijmans #  2 , Annemiek M C Gennissen #  2 , Nelson K Y Wong  1   3 , Shang Li  4 , G Bea A Wisman  5 , Aleksandra Hamilton  1 , Joshua Hoenisch  1 , Amy Dawson  1 , Cheng-Han Lee  1 , Madison Bittner  1 , Hannah Kim  1 , Gabriel E DiMattia  6 , Christianne A R Lok  7 , Cor Lieftink  2 , Roderick L Beijersbergen  2 , Steven de Jong  4 , Mark S Carey  1 , René Bernards  2 , Katrien Berns  2
Affiliations

NOTCH Signaling Limits the Response of Low-Grade Serous Ovarian Cancers to MEK Inhibition

Marta Llaurado Fernandez et al. Mol Cancer Ther. .

Abstract

Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian cancer with high fatality rates in advanced stages due to its chemoresistant properties. LGSOC is characterized by activation of MAPK signaling, and recent clinical trials indicate that the MEK inhibitor (MEKi) trametinib may be a good treatment option for a subset of patients. Understanding MEKi-resistance mechanisms and subsequent identification of rational drug combinations to suppress resistance may greatly improve LGSOC treatment strategies. Both gain-of-function and loss-of-function CRISPR-Cas9 genome-wide libraries were used to screen LGSOC cell lines to identify genes that modulate the response to MEKi. Overexpression of MAML2 and loss of MAP3K1 were identified, both leading to overexpression of the NOTCH target HES1, which has a causal role in this process as its knockdown reversed MEKi resistance. Interestingly, increased HES1 expression was also observed in selected spontaneous trametinib-resistant clones, next to activating MAP2K1 (MEK1) mutations. Subsequent trametinib synthetic lethality screens identified SHOC2 downregulation as being synthetic lethal with MEKis. Targeting SHOC2 with pan-RAF inhibitors (pan-RAFis) in combination with MEKi was effective in parental LGSOC cell lines, in MEKi-resistant derivatives, in primary ascites cultures from patients with LGSOC, and in LGSOC (cell line-derived and patient-derived) xenograft mouse models. We found that the combination of pan-RAFi with MEKi downregulated HES1 levels in trametinib-resistant cells, providing an explanation for the synergy that was observed. Combining MEKis with pan-RAFis may provide a promising treatment strategy for patients with LGSOC, which warrants further clinical validation.

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Figures

Figure 1. Genome-wide trametinib-resistance screen identifies MAML2 and MAP3K1. A, Schematic overview of genome-wide trametinib resistance screen in VOA-6406dCas9MS2 cell line with the SAM library targeting 23,430 isoforms for transcriptional activation. Cells were infected with MOI below 0.5 and 250× representation of guides. Trametinib selection was performed for 3 weeks, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit MAML2 is highlighted in the bubble plot presentation of the DESeq2/RRA MAGeCK analysis. FDR threshold <0.1. B, The functional phenotypes of lentiviral SAM1.2-gMAML2 vectors (#1 and #2) in the VOA-6406 dCas9MS2 cell line upon trametinib treatment was measured in a long-term colony-formation assay. Cells expressing an empty pSAM1.2 vector were used as control. The cells were fixed, stained, and scanned after 14 days. C, Western blot analysis of the VOA-6406 dCas9MS2 cell line transduced with lentiviral SAM1.2 and SAM1.2-gMAML2#2 and exposed to 0, 2, and 5 nmol/L trametinib for 3 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. D, VOA-6406dCas9MS2 cells transduced with SAM1.2 and SAM1.2gMAML2#2 were subjected to mRNA expression analysis with QRT-PCR. mRNA levels were normalized to the expression of GAPDH and displayed is the relative expression of the indicated mRNAs. Error bars denote SD. E, Schematic overview of genome-wide trametinib-resistance screen in VOA-6406 cell line with the Brunello library targeting 19,114 genes. Cells were infected with MOI below 0.5 and 250× representation of guides. Trametinib selection was performed for 3 weeks, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit MAP3K1 is highlighted in the bubble plot presentation of the DEseq2/RRA MAGeCK analysis. FDR threshold <0.1. F, Colony-formation assays were performed with selected VOA-6406 monoclonal cell lines transduced with gCTRL#1 (nontargeting region in chromosome 5), gCTRL#2 (nontargeting region in chromosome 9), and gMAP3K1#1 and gMAP3K1#2. Plated cells were exposed to increasing trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 10 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. G, Western blot analysis of total lysates generated from the monoclonal gCTRL (#1 and #2) and gMAP3K1 (#1 and #2) lines exposed to 0, 2, and 10 nmol/L trametinib for 1 day. Blots were probed with the indicated antibodies. H, Western blot analysis of the VOA-6406 dCas9MS2 cell line transduced with lentiviral SAM1.2 and SAM1.2-gMAML2#2 and the VOA-6406 monoclonal lines gCTRL (#1 and #2) and gMAP3K1 (#1 and #2). Blots were probed with the indicated antibodies.
Figure 1.
Genome-wide trametinib-resistance screen identifies MAML2 and MAP3K1.A, Overview of genome-wide trametinib resistance screen in VOA-6406dCas9MS2 cell line with the SAM library targeting 23,430 isoforms for transcriptional activation. Cells were infected with MOI below 0.5 and 250× representation of guides. Trametinib selection was performed for 3 weeks, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit MAML2 is highlighted in the bubble plot presentation of the DESeq2/ RRA MAGeCK analysis. FDR threshold <0.1. B, The functional phenotypes of lentiviral SAM1.2-gMAML2 vectors (No. 1 and No. 2) in the VOA-6406 dCas9MS2 cell line upon trametinib treatment was measured in a long-term colony-formation assay. Cells expressing an empty pSAM1.2 vector were used as control. The cells were fixed, stained, and scanned after 14 days. C, Western blot analysis of the VOA-6406 dCas9MS2 cell line transduced with lentiviral SAM1.2 and SAM1.2-gMAML2 No.2 and exposed to 0, 2, and 5 nmol/L trametinib for 3 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. D, VOA-6406dCas9MS2 cells transduced with SAM1.2 and SAM1.2gMAML2 No. 2 were subjected to mRNA expression analysis with QRT-PCR. mRNA levels were normalized to the expression of GAPDH and displayed is the relative expression of the indicated mRNAs. Error bars denote SD. E, Overview of genome-wide trametinib-resistance screen in VOA-6406 cell line with the Brunello library targeting 19,114 genes. Cells were infected with MOI below 0.5 and 250× representation of guides. Trametinib selection was performed for 3 weeks, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit MAP3K1 is highlighted in the bubble plot presentation of the DEseq2/ RRA MAGeCK analysis. FDR threshold <0.1. F, Colony-formation assays were performed with selected VOA-6406 monoclonal cell lines transduced with gCTRL No. 1 (nontargeting region in chromosome 5), gCTRL No. 2 (nontargeting region in chromosome 9), and gMAP3K1 No. 1 and gMAP3K1 No. 2. Plated cells were exposed to increasing trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 10 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. G, Western blot analysis of total lysates generated from the monoclonal gCTRL (No. 1 and No. 2) and gMAP3K1 (No. 1 and No. 2) lines exposed to 0, 2, and 10 nmol/L trametinib for 1 day. Blots were probed with the indicated antibodies. H, Western blot analysis of the VOA-6406 dCas9MS2 cell line transduced with lentiviral SAM1.2 and SAM1.2-gMAML2 No. 2 and the VOA-6406 monoclonal lines gCTRL (No. 1 and No. 2) and gMAP3K1 (No. 1 and No. 2). Blots were probed with the indicated antibodies.
Figure 2. Spontaneous trametinib resistance through MAP2K1 mutation or HES1 upregulation. A, VOA-6406 cells were plated at low density, and spontaneous trametinib-resistant clones were selected by 4–6 weeks of exposure to 5 or 20 nmol/L trametinib as indicated. Selected clones were subjected to targeted sequence analysis of 178 cancer-associated genes (NKI-178 panel). B, Western blot analysis of total lysates generated from the parental VOA-6406 (PAR) and the monoclonal VOA-6406 R#1, R#2, R#3, R#4, and R#5 lines exposed to 5 nmol/L trametinib for 2 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. C, The parental VOA-6406 (PAR) and monoclonal VOA-6406 R#1, R#2, R#3, R#4, and R#5 lines were subjected to mRNA expression analysis with QRT-PCR. mRNA levels of HES1 were normalized to the expression of GAPDH, and displayed is the relative expression in the indicated monoclonal trametinib-resistant lines. Error bars, SD. D, Colony-formation assays were performed with the VOA-6406-R#2 line, which was infected with shCTRL (scrambled hairpins) and shHES1 constructs. Stably selected cells were exposed to increasing trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 11 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. E, Western blot analysis of total lysates generated from the shCTRL- and shHES1-transduced VOA-6406 and VOA-6406-R#2 lines. Blots were probed with HES1 and HSP90 (loading control) antibodies. F, Crystal Violet quantification of colony-formation assays performed as described in D. Displayed are the relative values compared with untreated condition. Error bars, SD. G, Colony-formation assays were performed with VOA-6406-R#2 incubated in the presence of trametinib (0, 5, 10 nmol/L) and/or the HES inhibitor JI051 (25 nmol/L) as indicated. The cells were fixed, stained, and scanned after 10 days. H, CI were calculated for the trametinib/JI051 combination in parental VOA-6406 and VOA-6406-R#2. All 384-well growth assays were performed multiple times and were normalized to positive (POA) and negative (DMSO) controls. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. Values below 0.9 indicate synergy. I, Western blot analysis of total lysates generated from parental (PAR) and TRAM resistant (RES) subclones derived from iOvCa241 by prolonged TRAM (20 nmol/L) exposure. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. J, Parental and TRAM-resistant cell line pairs were plated at equal densities, and the next day total lysates were subjected to Western analysis with the indicated antibodies. K, Colony-formation assays were performed in VOA-6406-gCTRL#1 and VOA-6406-gMAP3K1#2 with increasing (combination) dosage of trametinib and the gamma-secretase inhibitor RO4929097 as indicated. The cells were fixed, stained, and scanned after 7 days. Colony-formation assays were performed in triplicate, and a representative staining is shown.
Figure 2.
Spontaneous trametinib resistance through MAP2K1 mutation or HES1 upregulation. A, VOA-6406 cells were plated at low density, and spontaneous trametinib-resistant clones were selected by 4 to 6 weeks of exposure to 5 or 20 nmol/L trametinib as indicated. Selected clones were subjected to targeted sequence analysis of 178 cancer-associated genes (NKI-178 panel). B, Western blot analysis of total lysates generated from the parental VOA-6406 (PAR) and the monoclonal VOA-6406 R No. 1, R No. 2, R No. 3, R No. 4, and R No. 5 lines exposed to 5 nmol/L trametinib for 2 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. C, The parental VOA-6406 (PAR) and monoclonal VOA-6406 R No. 1, R No. 2, R No. 3, R No. 4, and R No. 5 lines were subjected to mRNA expression analysis with QRT-PCR. mRNA levels of HES1 were normalized to the expression of GAPDH, and displayed is the relative expression in the indicated monoclonal trametinib-resistant lines. Error bars, SD. D, Colony-formation assays were performed with the VOA-6406-R No. 2 line, which was infected with shCTRL (scrambled hairpins) and shHES1 constructs. Stably selected cells were exposed to increasing trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 11 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. E, Western blot analysis of total lysates generated from the shCTRL- and shHES1-transduced VOA-6406 and VOA-6406-R No. 2 lines. Blots were probed with HES1 and HSP90 (loading control) antibodies. F, Crystal violet quantification of colony-formation assays performed as described in D. Displayed are the relative values compared with untreated condition. Error bars, SD. G, Colony-formation assays were performed with VOA-6406-R No. 2 incubated in the presence of trametinib (0, 5, 10 nmol/L) and/or the HES inhibitor JI051 (25 nmol/L) as indicated. The cells were fixed, stained, and scanned after 10 days. H, CI were calculated for the trametinib/JI051 combination in parental VOA-6406 and VOA-6406-R No. 2. All 384-well growth assays were performed multiple times and were normalized to positive (POA) and negative (DMSO) controls. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. Values below 0.9 indicate synergy. I, Western blot analysis of total lysates generated from parental (PAR) and TRAM-resistant (RES) subclones derived from iOvCa241 by prolonged TRAM (20 nmol/L) exposure. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. J, Parental and TRAM-resistant cell line pairs were plated at equal densities, and the next day, total lysates were subjected to Western analysis with the indicated antibodies. K, Colony-formation assays were performed in VOA-6406-gCTRL No. 1 and VOA-6406-gMAP3K1 No. 2 with increasing (combination) dosage of trametinib and the gamma-secretase inhibitor RO4929097 as indicated. The cells were fixed, stained, and scanned after 7 days. Colony-formation assays were performed in triplicate, and a representative staining is shown.
Figure 3. Genome-wide trametinib enhancer screen identifies SHOC2. A, Schematic overview of genome-wide trametinib enhancer screen in VOA-4627 cell line with the Brunello library targeting 19,114 genes. Cells were infected with MOI below 0.5 and 1,000× representation of guides. Trametinib selection was performed for 10 days, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit SHOC2 is highlighted in the bubble plot presentation of the RRA MAGeCK analysis. FDR threshold <0.1. B, Colony-formation assays were performed with VOA-4627 cells stably infected with shCTRL, shSHOC2#1, and shSHOC2#2. Plated cells were exposed to 2 nmol/L trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 10 days. C, Western blot analysis of total lysates generated from shCTRL, shSHOC2#1, and shSHOC2#2 VOA-4627 lines exposed to 0 and 10 nmol/L trametinib for 4 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. D, Cell proliferation assay (IncuCyte) was performed for the doxycycline (DOX) inducible shSHOC2 VOA-6406 cell line with the MEKi trametinib (TRAM 2 nmol/L) in the absence or presence of DOX 0.5 μg/mL. E, Western blot analysis of total lysates generated from the doxycycline-inducible shSHOC2 VOA-6406 cell line (without and with DOX 0.5 μg/mL) exposed to 0 and 2 nmol/L trametinib for 2 days. Blots were probed with the indicated antibodies. ACTIN was used as a loading control. F, Cell viability (crystal violet) assay was performed with a doxycycline-inducible shSHOC2 VOA-6406 cell line (without and with DOX 0.5 μg/mL) in the presence of the indicated drugs; MEKi trametinib (TRAM, 2 nmol/L), pan-RAF inhibitor (LY3009120, 10 nmol/L) after 4 days of drug exposure. Crystal violet measurements were normalized to their respective DMSO controls. G, Colony-formation assays were performed with VOA-3723, VOA-4627, VOA-6406, VOA-6406-R#1, VOA-6406-R#2, and VOA-6406-R#5 with increasing (combination) dosage of trametinib and the pan-RAFi LY3009120 as indicated. The cells were fixed, stained, and scanned after 7 days. H, CI were calculated for the trametinib/LY3009120 combination in a 384-well format normalized to positive (POA) and negative (DMSO) controls. All 384-well growth assays were plated in quadruple and performed multiple times. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. CI scores are defined as: <0.1 very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.7–0.85 moderate synergism; and 0.85–0.9 slight synergism.
Figure 3.
Genome-wide trametinib enhancer screen identifies SHOC2.A, Overview of genome-wide trametinib enhancer screen in VOA-4627 cell line with the Brunello library targeting 19,114 genes. Cells were infected with MOI below 0.5 and 1,000× representation of guides. Trametinib selection was performed for 10 days, after which gRNA abundance was determined in treated (tr) over untreated (ut) conditions through deep sequencing. Screens were performed in triplicate. Validated hit SHOC2 is highlighted in the bubble plot presentation of the RRA MAGeCK analysis. FDR threshold <0.1. B, Colony-formation assays were performed with VOA-4627 cells stably infected with shCTRL, shSHOC2 No. 1, and shSHOC2 No. 2. Plated cells were exposed to 2 nmol/L trametinib concentrations as indicated. The cells were fixed, stained, and scanned after 10 days. C, Western blot analysis of total lysates generated from shCTRL, shSHOC2 No. 1, and shSHOC2 No. 2 VOA-4627 lines exposed to 0 and 10 nmol/L trametinib for 4 days. Blots were probed with the indicated antibodies. HSP90 was used as a loading control. D, Cell proliferation assay (IncuCyte) was performed for the doxycycline (DOX) inducible shSHOC2 VOA-6406 cell line with the MEKi trametinib (TRAM 2 nmol/L) in the absence or presence of DOX 0.5 μg/mL. E, Western blot analysis of total lysates generated from the doxycycline-inducible shSHOC2 VOA-6406 cell line (without and with DOX 0.5 μg/mL) exposed to 0 and 2 nmol/L trametinib for 2 days. Blots were probed with the indicated antibodies. ACTIN was used as a loading control. F, Cell viability (crystal violet) assay was performed with a doxycycline-inducible shSHOC2 VOA-6406 cell line (without and with DOX 0.5 μg/mL) in the presence of the indicated drugs; MEKi trametinib (TRAM, 2 nmol/L), pan-RAF inhibitor (LY3009120, 10 nmol/L) after 4 days of drug exposure. Crystal violet measurements were normalized to their respective DMSO controls. G, Colony-formation assays were performed with VOA-3723, VOA-4627, VOA-6406, VOA-6406-R No. 1, VOA-6406-R No. 2, and VOA-6406-R No. 5 with increasing (combination) dosage of trametinib and the pan-RAFi LY3009120 as indicated. The cells were fixed, stained, and scanned after 7 days. H, CIs were calculated for the trametinib/LY3009120 combination in a 384-well format normalized to positive (POA) and negative (DMSO) controls. All 384-well growth assays were plated in quadruple and performed multiple times. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. CI scores are defined as <0.1 very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.7–0.85 moderate synergism; and 0.85–0.9 slight synergism.
Figure 4. MEKi/pan-RAFi synergy with the pan-RAFi BGB283. Colony-formation assays and CI calculations were performed for the cell line pairs VOA-6406-PAR and VOA-6406-R#2 (A, B), iOvCa241-PAR and iOvCa241-RES (C, D), and VOA-6406-gCTRL#1 and VOA-6406-gMAP3K1#2 (E, F) with increasing (combination) dosage of trametinib and the pan-RAFi BGB283 as indicated. The cells were fixed, stained, and scanned after 7 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. CI were calculated for the trametinib/BGB283 combination in 384-well format normalized to positive (POA) and negative (DMSO) controls. All 384-well growth assays were plated in quadruple and performed multiple times. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. CI scores are defined as: <0.1 very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.7–0.85 moderate synergism; and 0.85–0.9 slight synergism. G, Colony formation washout experiment with VOA-6406. Cells were exposed to the indicated drugs for 1 week, after which cells were washed and cultured in a normal medium without drugs for 3 weeks. Cells were fixed, stained, and scanned. H and I, Western blot analyses of total lysates generated from the parental and TRAM-resistant cell line pairs iOvCa241-PAR/RES (G) VOA-6406-PAR/VOA-6406-R#2 and VOA-6406 gCTRL#1/gMAP3K1#2 (H) exposed to 1 μmol/L BGB283 (B), 10 nmol/L trametinib (T) or the combination (B/T). Blots were probed with the indicated antibodies.
Figure 4.
MEKi/pan-RAFi synergy with the pan-RAFi BGB283. Colony-formation assays and CI calculations were performed for the cell line pairs VOA-6406-PAR and VOA-6406-R No. 2 (A, B), iOvCa241-PAR and iOvCa241-RES (C, D), and VOA-6406-gCTRL No. 1 and VOA-6406-gMAP3K1 No. 2 (E, F) with increasing (combination) dosage of trametinib and the pan-RAFi BGB283 as indicated. The cells were fixed, stained, and scanned after 7 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. CIs were calculated for the trametinib/BGB283 combination in 384-well format normalized to positive (POA) and negative (DMSO) controls. All 384-well growth assays were plated in quadruple and performed multiple times. Displayed are the median CI values of the synergy 5 × 5 concentration matrix. Error bars, SD. CI scores are defined as <0.1 very strong synergism; 0.1–0.3 strong synergism; 0.3–0.7 synergism; 0.7–0.85 moderate synergism; and 0.85–0.9 slight synergism. G, Colony formation washout experiment with VOA-6406. Cells were exposed to the indicated drugs for 1 week, after which cells were washed and cultured in a normal medium without drugs for 3 weeks. Cells were fixed, stained, and scanned. H and I, Western blot analyses of total lysates generated from the parental and TRAM-resistant cell line pairs iOvCa241-PAR/RES (H) VOA-6406-PAR/VOA-6406-R No. 2 and VOA-6406 gCTRL No. 1/gMAP3K1 No. 2 (I) exposed to 1 μmol/L BGB283 (B), 10 nmol/L trametinib (T) or the combination (B/T). Blots were probed with the indicated antibodies.
Figure 5. MEKi/pan-RAFi synergy in primary ascites cultures. A, Ascites was retrieved from chemo-naïve patients with LGSOC during debulking surgery or procedures aimed to relieve clinical symptoms. Fresh ascites was directly put in culture flasks yielding a primary ascites culture after 1–2 weeks with medium replacement every 3 days. B, Western blot analysis of lysates generated from the primary ascites cultures as indicated. Blots were probed with PAX8 or CK7 antibodies. HSP90 was used as a loading control. The 293 cell lysate was used as a negative control. C, Long-term colony-formation assays were performed with the primary ascites cultures KAM003A, KAM004A, and KAM005A with increasing (combination) dosage of MEKi trametinib and the pan-RAFi LY3009120 as indicated. The cells were fixed, stained, and scanned after 10 days. D, Pictures of the primary ascites cultures KAM003A and KAM004A with indicated (combination) dosage of MEKi trametinib and the pan-RAFi LY3009120 after 10 days. E, Western blot of the KAM400A cells infected with shSCR or shSHOC2 are probed with the indicated antibodies. F, Long-term colony-formation assay was performed with KAM010A with increasing (combination) dosage of MEKi trametinib and pan-RAFi BGB283 as indicated. The cells were fixed, stained, and scanned after 10 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. G, KAM010A cells were exposed to 1 μmol/L BGB283 (B), 10 nmol/L trametinib (T) or the combination (B/T) for 2 days, after which lysates were analyzed by Western blot with the indicated antibodies.
Figure 5.
MEKi/pan-RAFi synergy in primary ascites cultures. A, Ascites was retrieved from chemo-naïve patients with LGSOC during debulking surgery or procedures aimed to relieve clinical symptoms. Fresh ascites was directly put in culture flasks yielding a primary ascites culture after 1 to 2 weeks with medium replacement every 3 days. B, Western blot analysis of lysates generated from the primary ascites cultures as indicated. Blots were probed with PAX8 or CK7 antibodies. HSP90 was used as a loading control. The 293 cell lysate was used as a negative control. C, Long-term colony-formation assays were performed with the primary ascites cultures KAM003A, KAM004A, and KAM005A with increasing (combination) dosage of MEKi trametinib and the pan-RAFi LY3009120 as indicated. The cells were fixed, stained, and scanned after 10 days. D, Pictures of the primary ascites cultures KAM003A and KAM004A with indicated (combination) dosage of MEKi trametinib and the pan-RAFi LY3009120 after 10 days. E, Western blot of the KAM400A cells infected with shSCR or shSHOC2 are probed with the indicated antibodies. F, Long-term colony-formation assay was performed with KAM010A with increasing (combination) dosage of MEKi trametinib and pan-RAFi BGB283 as indicated. The cells were fixed, stained, and scanned after 10 days. Colony-formation assays were performed in triplicate, and a representative staining is shown. G, KAM010A cells were exposed to 1 μmol/L BGB283 (B), 10 nmol/L trametinib (T) or the combination (B/T) for 2 days, after which lysates were analyzed byWestern blot with the indicated antibodies.
Figure 6. MEKi/pan-RAFi in the VOA-6406 xenograft and LGSOC PDX mouse model. A, VOA-6406 tumors were implanted in NRG mice and treatments (BGB283 5 or 10 mg/kg, trametinib (TRAM) 0.5 mg/kg, BGB283 5 mg/kg + trametinib 0.5 mg/kg) were started when tumors reached 120 mm3. Shown are relative tumor volumes; error bars, SEM. B, Tumor volumes were measured at day 33, and changes in tumor volume compared with baseline (day 0) are indicated. C, Relative tumor volumes of the individual tumors of the slow-growing LGSOC PDX model OC.79 (RASG13D-mutant), under the indicated treatments. Treatments were stopped when tumors reached maximum volume (1,500 mm3) or after 57 days. D, Tumor volume changes at the endpoint compared with the start of treatment for the LGSOC PDX model OC.79. E, Survival curve for the LGSOC PDX model OC.79.
Figure 6.
MEKi/pan-RAFi in the VOA-6406 xenograft and LGSOC PDX mouse model. A, VOA-6406 tumors were implanted in NRG mice and treatments (BGB283 5 or 10 mg/kg, trametinib (TRAM) 0.5 mg/kg, BGB283 5 mg/kg + trametinib 0.5 mg/kg) were started when tumors reached 120 mm3. Shown are relative tumor volumes; error bars, SEM. B, Tumor volumes were measured at day 33, and changes in tumor volume compared with baseline (day 0) are indicated. C, Relative tumor volumes of the individual tumors of the slow-growing LGSOC PDX model OC.79 (RASG13D-mutant), under the indicated treatments. Treatments were stopped when tumors reached maximum volume (1,500 mm3) or after 57 days. D, Tumor volume changes at the endpoint compared with the start of treatment for the LGSOC PDX model OC.79. E, Survival curve for the LGSOC PDX model OC.79.
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References

    1. Gershenson DM. Low-grade serous carcinoma of the ovary or peritoneum. Ann Oncol 2016;27(suppl 1):i45–i49. - PubMed
    1. Singer G, Oldt R, 3rd, Cohen Y, Wang BG, Sidransky D, Kurman RJ, et al. . Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003;95:484–6. - PubMed
    1. Farley J, Brady WE, Vathipadiekal V, Lankes HA, Coleman R, Morgan MA, et al. . Selumetinib in women with recurrent low-grade serous carcinoma of the ovary or peritoneum: an open-label, single-arm, phase 2 study. Lancet Oncol 2013;14:134–40. - PMC - PubMed
    1. Gershenson DM, Sun CC, Bodurka D, Coleman RL, Lu KH, Sood AK, et al. . Recurrent low-grade serous ovarian carcinoma is relatively chemoresistant. Gynecol Oncol 2009;114:48–52. - PubMed
    1. Monk BJ, Grisham RN, Banerjee S, Kalbacher E, Mirza MR, Romero I, et al. . MILO/ENGOT-ov11: binimetinib versus physician's choice chemotherapy in recurrent or persistent low-grade serous carcinomas of the ovary, fallopian tube, or primary peritoneum. J Clin Oncol 2020;38:3753–62. - PMC - PubMed

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