Subtype-specific genomic alterations define new targets for soft tissue sarcoma therapy

Authors

Jordi Barretina

Barry S. Taylor

Shantanu Banerji

Alexis H. Ramos

Mariana Lagos-Quintana

Penelope L. DeCarolis

Cristina R. Antonescu

Marc Ladanyi

Chris Sander

Matthew Meyerson

Samuel Singer

Doi

10.1038/ng.619

PMID: 20601955 · DOI: 10.1038/ng.619 · Journal: Nature Genetics (2010)

TL;DR

Barretina et al. performed integrative DNA sequencing (722 protein-coding and microRNA genes), 250K SNP-array copy-number profiling, and gene-expression analysis on 207 high-grade soft tissue sarcomas across seven major subtypes from the MSKCC Sarcoma Genome Project (sarc_mskcc). They identified subtype-specific drivers — TP53 in pleomorphic liposarcoma, NF1 in myxofibrosarcoma and pleomorphic liposarcoma, and PIK3CA in myxoid/round-cell liposarcoma — and used a shRNA screen in dedifferentiated liposarcoma cell lines to nominate CDK4, MDM2, and YEATS4 on the recurrent 12q amplicon as therapeutic targets.

Cohort & data

  • 207 patients with high-grade soft tissue sarcoma, seven subtypes: DDLS (n=50, 24.2%), MRLS (n=21, 10.1%), PLLS (n=24, 11.6%), LMS (n=27, 13%), GIST (n=22, 10.6%, epithelioid + spindle + mixed), MFS (n=38, 18.4% incl. pleomorphic MFH), SYNS (n=24, 11.6%).
  • Dataset: sarc_mskcc; raw data deposited in NCBI GEO under accession GSE21124.
  • Assays: targeted resequencing of 722 genes on 47 tumor/normal pairs, extended genotyping on 160 additional tumors via sequenom-genotyping; somatic copy-number alterations via 250K SNP arrays (affymetrix-250k-snp-array, n=207); LOH (n=200); oligonucleotide expression arrays (n=149); GISTIC + RAE for SCNA significance (gistic); functional validation via shrna-rnai-screen of 385 genes (2,007 shRNAs, median 5 per gene) in three DDLS cell lines (LPS141, DDLS8817, FU-DDLS-1).

Key findings

  • TP53 in PLLS: TP53 was mutated in 17% of pleomorphic liposarcomas — the only subtype in which TP53 mutations were detected in this cohort.
  • NF1 in MFS and PLLS: NF1 was mutated in 10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas, with six point mutations and twelve genomic deletions across the cohort. Two mutations (R304, Q369) were previously reported as germline NF1 alleles. Biallelic inactivation (heterozygous point mutation + WT-allele deletion) was evident in some tumors with correspondingly reduced expression vs. normal adipose (ANOVA p=1.94×10⁻⁵). First report of somatic NF1 alterations outside MPNST/GIST in NF1 patients.
  • PIK3CA in MRLS: PIK3CA was mutated in 18% (13/71) of myxoid/round-cell liposarcomas (initial 4/21 + 9 additional from a 50-patient validation cohort). Mutations clustered in helical (E542K, E545K) and kinase (H1047L, H1047R) domains. Mutated tumors had shorter disease-specific survival than WT (log-rank p=0.036, n=65 with outcome data); helical-domain mutants vs. WT p=0.013.
  • PIK3CA mechanism: Only E545K helical-domain mutants showed increased Akt phosphorylation (Ser473 / Thr308) and increased phospho-PRAS40 / phospho-S6K relative to WT. H1047R kinase-domain tumors did not show comparable AKT activation, possibly explained by variably elevated PTEN. One MRC tumor had homozygous PTEN deletion with high phospho-AKT.
  • KIT and PDGFRA: KIT was frequently mutated in GIST (and unexpectedly in one MRC sample); PDGFRA and KIT in GIST recapitulate known biology motivating imatinib therapy.
  • 12q amplification in DDLS: Chromosome 12q amplification was the most prevalent SCNA in any subtype, occurring in ~90% of dedifferentiated liposarcomas.
  • Functional shRNA screen (DDLS): Of 385 genes screened, 99 had significant anti-proliferative effects in ≥1 of 3 DDLS cell lines (nominal p<0.05); 91/99 confirmed by ≥2 independent shRNAs. Only 1 of 58 overlapping genes (PSMB4) was a common essential gene in a 12-cell-line pan-cancer reference screen — supporting DDLS-specific dependencies. 27 of the 99 genes were amplified in ≥1 DDLS line.
  • CDK4 dependency: CDK4 was the most overexpressed of the 27 amplified hits relative to normal fat. Sustained CDK4 shRNA knockdown (>10 days) inhibited proliferation in LPS141 and DDLS8817; pharmacologic CDK4/CDK6 inhibition with PD0332991 (palbociclib) induced G1 arrest in both lines.
  • MDM2 + YEATS4 cooperativity: MDM2 shRNA knockdown impaired proliferation only with sustained (>1 week) depletion. YEATS4 (GAS41), frequently co-amplified with MDM2 on 12q, was upregulated in tumors vs. normal fat and in amplified vs. copy-neutral tumors; YEATS4 knockdown phenocopied the screen result, supporting cooperative repression of the p53 network in DDLS.
  • Karyotypic complexity: MRLS, SYNS, and GIST had relatively normal karyotypes; DDLS, PLLS, LMS, and MFS were complex. Copy-neutral LOH was confined to the four complex subtypes. PLLS and MFS were strikingly similar genomically, supporting a unified molecular class.

Genes & alterations

  • TP53 — point mutations in 17% PLLS; restricted to this subtype in this cohort.
  • NF1 — point mutations + genomic deletions; 10.5% MFS, 8% PLLS; biallelic inactivation observed; loss linked to mTOR pathway activation.
  • PIK3CA — helical (E542K, E545K) and kinase (H1047L, H1047R) hotspots in 18% MRLS; helical mutations associated with AKT activation and worse DSS.
  • PTEN — homozygous deletion in one MRC tumor; splice-site mutation in PLLS.
  • CDK4 — focal 12q amplification in DDLS; functionally validated dependency; targetable by palbociclib.
  • MDM2 — focal 12q amplification in DDLS; sustained-knockdown sensitive; rationale for p53–MDM2 antagonists (nutlin-3a).
  • YEATS4 — co-amplified with MDM2 on 12q; knockdown anti-proliferative; proposed p53-network repressor.
  • KIT — recurrent activating mutations in GIST; one MRC sample.
  • PDGFRA — known GIST driver (background; motivates imatinib precedent).
  • CDH1 — resequenced in DDLS; one mutation found.
  • ERBB4 — resequenced in PLLS/MFS; mutations found.
  • RB1 — mutation in PLLS; broader Rb pathway implication consistent with CDK4 dependency.
  • AKT1 — pathway readout (phospho-Akt) in PIK3CA-mutant MRC; not somatically mutated in this study.

Clinical implications

  • PI3K inhibitors for MRLS: PIK3CA mutations identify a subset of MRLS potentially responsive to PI3K inhibitors then in clinical trials — first report of PIK3CA mutation in a mesenchymal cancer.
  • CDK4/CDK6 inhibitors for DDLS: Functional and pharmacologic data with PD0332991 (palbociclib) provide rationale for CDK4 inhibition in DDLS.
  • mTOR inhibitors for NF1-deficient sarcomas: Authors propose mTOR inhibition for NF1-loss MFS and PLLS, since NF1 loss activates mTOR.
  • MDM2 antagonists for DDLS: Co-amplification of MDM2 + YEATS4 supports clinical evaluation of p53–MDM2 interaction antagonists (nutlin-3a) in DDLS.
  • Genotype-directed precedent: The work explicitly invokes the GIST/KIT → imatinib paradigm as the model for subtype-specific sarcoma therapy.

Limitations & open questions

  • Subtype cohorts are modest (e.g., 21 MRLS in discovery); the helical- vs. kinase-domain DSS difference did not reach statistical significance.
  • Functional impact of singleton mutations across many genes (Table 2) was not established and “further studies will be needed.”
  • Relationship between PI3K activation from PIK3CA mutation and the pathognomonic t(12;16)(q13;p11) FUS–DDIT3 translocation in MRLS remains unresolved.
  • For most candidate dependencies, validation was in cell lines — patient-level efficacy of CDK4 or MDM2 antagonists awaits trials.
  • 722-gene targeted panel: many cancer-relevant loci outside the panel were not interrogated; whole-exome/whole-genome views of these subtypes were left to future work.

Citations from this paper used in the wiki

  • “PIK3CA, in 18% of myxoid/round cell liposarcomas, TP53 in 17% of pleomorphic liposarcomas (interestingly, the only subtype in which mutations of this gene were found), and NF1 in 10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas” (p. 3).
  • “MRC patients whose tumors harbored mutations in PIK3CA had a shorter duration of disease–specific survival than did those with wildtype PIK3CA (p=0.036, log-rank test)” (p. 4).
  • “The alteration with the highest prevalence in any subtype was chromosome 12q amplification in dedifferentiated liposarcoma (~90%)” (p. 5).
  • “pharmacological inhibition of CDK4 in dedifferentiated liposarcoma cells with PD0332991, a selective CDK4/CDK6 inhibitor currently in clinical trials, induced G1 arrest” (p. 5–6).
  • “YEATS4 and MDM2 amplification may cooperatively repress the p53 network in dedifferentiated liposarcoma” (p. 6).
  • Cohort composition (Table 1): 207 patients across seven subtypes, MSKCC.

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