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JULY 2014�CANCER DISCOVERY | 745

IN THIS ISSUEIN THIS ISSUE

Dissemination of cancer cells

from primary tumors has been

shown to occur early during

tumor growth in both breast and

pancreatic cancer, suggesting that

these cancer cells already possess

the genetic changes required

to initiate the metastatic cas-

cade. However, it is not known

whether early premalignant lung

lesions are also inherently capable of generating disseminated

tumor cells (DTC). To analyze lung cancer cell dissemination,

Caswell and colleagues utilized a genetically engineered mouse

model of lung cancer driven by Kras mutation in which tumor

cells were fl uorescently labeled. Intriguingly, few DTCs were

detected in Kras-mutant mice with either early-stage hyper-

plastic lesions or adenocarcinomas. Concomitant loss of p53

was associated with the presence of DTCs in a fraction of mice

with late-stage lung tumors, suggesting that p53 inactivation

is not suffi cient to facilitate dissemination but enables the

acquisition of additional genetic changes necessary to trigger

dissemination. These DTCs originated from a single primary

tumor, indicative of cell-autonomous alterations that confer

the ability to disseminate. In addition, DTCs exhibited reduced

expression of the prodifferentiation transcription factor Nkx2-

1, which was also downregulated in a portion of the parental

tumor cells coincident with increased proliferative potential,

supporting the notion that loss of Nkx2-1 in a subpopulation

of primary tumor cells promotes the generation of DTCs.

These results suggest that dissemination is an acquired phe-

notype in lung adenocarcinoma and a rate-limiting barrier to

lung cancer metastasis. ■

See article, p. 781.

• Kras-mutant, p53-deficient tumor

cells are not inherently capable of

dissemination in mice.

• p53 loss is insufficient to drive dis-

semination but enables acquisition

of required alterations.

• Nkx2-1 downregulation and

enhanced proliferation in primary

tumors precede dissemination.

Dissemination Is a Barrier to Lung Cancer Metastasis

Autophagy has been implicated

in tumorigenesis given its ability

to promote cancer cell survival

during stress, and the activating

BRAF V600E mutation is known to

enhance basal autophagic activ-

ity in diverse tumor types. The

BRAF V600E mutation has recently

been identifi ed in pediatric central

nervous system (CNS) tumors,

but whether autophagy contributes to BRAF V600E-induced

tumorigenesis in these tumor types is not fully understood.

Mulcahy Levy and colleagues found that BRAF V600E-positive

CNS tumor cell lines exhibited greater starvation-induced

autophagy than did BRAF–wild-type CNS tumor cells, raising

the possibility that the increased autophagy associated with

BRAF V600E might contribute a selective advantage to CNS

tumor cells. Indeed, genetic or pharmacologic inhibition of

autophagy was cytotoxic to BRAF-mutant CNS tumor cells,

but had a minimal effect on survival of BRAF–wild-type cells,

indicating that BRAF-mutant CNS tumor cells are depend-

ent on autophagy for survival. Moreover, treatment with the

autophagy inhibitor chloroquine improved the effectiveness

of both standard chemotherapeutics and the BRAF inhibitor

vemurafenib, and showed synergistic activity with vemuraf-

enib in BRAF-mutant CNS tumor cells at clinically achievable

doses. Of note, combined chloroquine and vemurafenib treat-

ment overcame vemurafenib resistance in primary BRAF V600E-

positive pleomorphic xanthoastrocytoma cells and led to rapid

clinical improvement and stabilization of disease in a patient

with vemurafenib-refractory BRAF V600E-positive brainstem

ganglioglioma that reversed whenever vemurafenib was discon-

tinued. These fi ndings indicating that BRAF-mutant pediatric

CNS tumors are autophagy-dependent provide a rationale for

combining autophagy inhibitors with BRAF-targeted therapy

in patients with relapsed or refractory disease. ■


• Genetic or pharmacologic inhibition

of autophagy is cytotoxic to

BRAFV600E-positive CNS tumor cells.

• Chloroquine synergizes with vemu-

rafenib or standard chemotherapy

in BRAF-mutant CNS tumor cells.

• A child with relapsed BRAF-mutant

ganglioglioma responded to chloro-

quine plus vemurafenib therapy.

Autophagy Inhibition Targets BRAF-Mutant Pediatric CNS Tumors

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IN THIS ISSUE

Dormant residual breast can-

cer cells often persist for long

periods following treatment and

can give rise to incurable recur-

rent tumors, emphasizing the

need to understand the molecu-

lar mechanisms that regulate the

outgrowth of these cells. Using

genetically engineered mouse

models, Feng and colleagues

found that SplA/ryanodine receptor domain and SOCS box

containing 1 (SPSB1) is upregulated in recurrent mammary

tumors and was both necessary and suffi cient for tumor recur-

rence following suppression of the driving HER2/neu onco-

gene. SPSB1 expression protected both murine and human

mammary tumor cells from apoptosis in response to HER2/

neu inhibition or treatment with chemotherapeutic agents

and was selected for during tumor outgrowth, indicating that

SPSB1 confers a growth advantage in residual breast cancer

cells. The prosurvival function of SPSB1 was dependent on

binding of SPSB1 to c-MET and potentiation of c-MET activity

in the absence of HER2/neu expression, as inhibition of c-MET

diminished breast cancer cell viability and prevented selection

of SPSB1-expressing cells in tumor-bearing mice. Furthermore,

elevated SPSB1 expression was associated with basal-like breast

cancer and was independently correlated with increased risk of

relapse only in patients with increased c-MET expression and

activity, suggesting that SPSB1 may contribute to the aggres-

sive phenotype and therapeutic resistance in these tumors via

activation of c-MET signaling. These fi ndings defi ne a role for

SPSB1-driven c-MET activity in breast cancer recurrence and

suggest that targeting SPSB1 may limit tumor relapse. ■


• SPSB1 upregulation is necessary

and sufficient for breast cancer

recurrence in mouse models.

• SPSB1 protects breast cancer cells

from apoptosis following HER2

inhibition or chemotherapy.

• Potentiation of c-MET signaling is

required for SPSB1-driven tumor

cell survival and recurrence.

SPSB1-Mediated c-MET Activation Drives Breast Cancer Recurrence

Women with a family history

of breast cancer have a 2- to 3-fold

higher risk of developing the

disease. Currently, only approxi-

mately 50% of familial breast

cancers can be attributed to

mutations in known cancer sus-

ceptibility genes, such as BRCA1

or BRCA2, which suggests that

additional genetic mutations

may confer hereditary breast cancer risk. Park and colleagues

used whole-exome sequencing to screen for previously unrec-

ognized cancer susceptibility genes in a cohort of 89 women

with early-onset breast cancer from highly selected families

with multiple cases of breast cancer. This approach iden-

tifi ed three separate, family-specifi c mutations in RAD50-

interacting protein 1 (RINT1) that were not observed in public

databases. In line with these fi ndings, case–control mutation

screening showed an enrichment of RINT1 variants that were

predicted to be deleterious in women with early-onset breast

cancer compared with age-matched controls, and an addi-

tional 4 RINT1 mutations were identifi ed in an independent

large cohort of multicase breast cancer families. Variants in

RINT1 that were likely to be pathogenic included missense

mutations, in-frame deletions, and mutations predicted to

affect RINT1 splicing. Of note, comparisons of cancer inci-

dence across RINT1 mutation–positive families revealed a sig-

nifi cantly higher risk for Lynch Syndrome–spectrum cancers

that are associated with DNA mismatch repair defects. These

fi ndings implicating RINT1 as a breast cancer susceptibility

gene suggest that RINT1 should be added to the list of genes

evaluated in genetic testing for hereditary breast cancer. ■


• RAD50-interacting protein 1 (RINT1)

mutations were identified in women from

multiple-case breast cancer families.

• Rare RINT1 variants were enriched

in early-onset breast cancer cases com-

pared with unaffected female controls.

• Carriers of RINT1 mutations also

display a higher incidence of Lynch

Syndrome–spectrum cancers.

RINT1 Is a Breast Cancer Predisposition Gene



JULY 2014�CANCER DISCOVERY | 747

IN THIS ISSUE

Inhibitors targeting the MAPK

pathway are clinically effective

in the majority of patients with

BRAF-mutant melanoma; how-

ever, a subset of patients fails to

respond due to intrinsic resist-

ance mechanisms that remain

poorly understood. Koniecz-

kowski and colleagues found

that MAPK inhibitor–sensitive

and intrinsically resistant melanoma cell lines and tumors

were defi ned by distinct and reciprocal gene expression pro-

fi les, suggesting that cell-autonomous differences contrib-

ute to RAF and MEK inhibitor drug resistance. Specifi cally,

intrinsically resistant melanomas were characterized by

low expression and activity of microphthalmia-associated

transcription factor (MITF) and elevated NF-κB pathway

signaling (MITF-low/NF-κB–high), whereas MAPK inhibi-

tor–sensitive melanomas were classifi ed as MITF-high/

NF-κB–low. Establishment of these two transcriptional

states in melanocytes was regulated by the balance between

oncogenic MAPK signaling, which activated NF-κB, and

sustained MITF expression. Stimulation of NF-κB activity

in MAPK inhibitor–sensitive cells induced a transition to

the MITF-low/NF-κB–high phenotype by suppressing MITF

and was suffi cient to confer resistance to MAPK pathway

inhibitors, indicative of plasticity between these states. Fur-

thermore, inhibition of BRAF in MITF-high, drug-sensitive

cells was associated with a transition to the MITF-low/

NF-κB–high state, suggesting that this phenotype correlates

with reduced dependence on MAPK signaling and may also

contribute to acquired MAPK inhibitor resistance. These

results identify transcriptional states that may underlie

intrinsic resistance to MAPK inhibition and may help to

predict therapeutic responses among patients with BRAF-

mutant melanoma. ■


• Reciprocal MITF and NF-κB activity

defines MAPK inhibitor–sensitive

and resistant melanomas.

• High NF-κB activity suppresses

MITF and confers intrinsic resis-

tance to MAPK inhibitors.

• Transition to an MITF-low/NF-κB–

high state may contribute to ac-

quired MAPK inhibitor resistance.

Transcription States Are Linked to Intrinsic Drug Resistance in Melanoma

Several cancers such as

gliomas and acute myeloid

leukemias harbor recurrent neo-

morphic mutations in isocitrate

dehydrogenase 1 or 2 (IDH1/2)

that cause the enzyme to con-

vert α-ketoglutarate (αKG) to

2-hydroxyglutarate (2HG), an

oncometabolite that modulates

αKG-dependent DNA and his-

tone demethylases and promotes cellular transformation.

To identify drugs that can reduce 2HG production, Elham-

mali and colleagues screened a library of compounds using

a high-throughput fl uorimetric assay that measured 2HG

levels in IDH1-mutant cells. The most potent compound

identifi ed by the screen was Zaprinast, a known inhibitor

of phosphodiesterase type 5 (PDE5). However, Zaprinast

did not reduce 2HG levels through inhibition of PDE5 but

instead acted through an off-target effect on glutaminase,

an enzyme that operates upstream of mutant IDH. Zap-

rinast noncompetitively inhibited glutaminase, preventing

the enzyme from metabolizing glutamine into glutamate,

a precursor of the mutant IDH substrate αKG. Accord-

ingly, Zaprinast reduced DNA and histone methylation

and prevented soft-agar colony formation in IDH1-mutant

cells, suggesting that this compound could reverse the

mutant IDH phenotype. Additionally, Zaprinast increased

levels of reactive oxygen species, enhanced susceptibility to

oxidative damage, and reduced growth in pancreatic ductal

adenocarcinoma cells that are dependent upon glutamine

metabolism. Although clinically useful dosages of Zapri-

nast may not be achievable given its higher potency against

PDE5 than glutaminase, these fi ndings raise the possibility

that Zaprinast or more glutaminase-selective Zaprinast

derivatives may have activity in IDH-mutant or glutamine-

addicted cancers. ■


• A fluorimetric assay was used in a

screen for drugs that reduce cellu-

lar levels of 2-hydroxyglutarate.

• Zaprinast blocks 2-hydroxygluta-

rate production through noncom-

petitive inhibition of glutaminase.

• IDH1-mutant and glutamine-ad-

dicted cancer cells are both sensi-

tive to Zaprinast.

Zaprinast Blocks 2-Hydroxyglutarate Production by Inhibiting Glutaminase



748 | CANCER DISCOVERY�JULY 2014 www.aacrjournals.org

IN THIS ISSUE

Disruption of the tumor sup-

pressor gene RB1 conforms to

the classical two-hit model, in

which mutation followed by

LOH promotes tumor forma-

tion. In addition to its role in

cell-cycle progression, the RB

protein (pRB) has also been sug-

gested to participate in DNA

replication, DNA repair, and

chromosome condensation, but the mechanism by which

pRB regulates genome stability remains unclear. Coschi and

colleagues found that loss of pRB or expression of a mutant

pRB enhanced γH2AX foci formation and triggered aberrant

DNA replication, particularly at major satellite repeats within

pericentromeric chromatin, indicative of replication stress.

Replication of pericentromeres was regulated by formation

of a complex between pRB, E2F1, and condensin II at major

satellite repeats. Intriguingly, loss of a single Rb1 allele reduced

condensin II recruitment to pericentromeres, induced γH2AX

deposition to a level similar to that of Rb1-defi cient cells, and

resulted in mitotic errors and chromosome structure defects,

suggesting that Rb1 is haploinsuffi cient for maintenance

of genome stability. Consistent with this idea, γH2AX foci

and mitotic defects were also enhanced in normal RB1+/−

fi broblasts from patients with hereditary retinoblastoma,

and RB1+/− cancer cell lines of mesenchymal origin harbored

increased chromosomal abnormalities, similar to RB1−/− cells.

Furthermore, tumors isolated from mice heterozygous for

mutant Rb1 exhibited increased chromosomal gains and

losses. These results identify a gene dosage–dependent func-

tion of pRB in suppressing genome instability and suggest

that disruption of this function contributes to aneuploidy

in cancer. ■


• A pRB–E2F1–condensin II complex

regulates DNA replication at peri-

centromeric chromatin.

• Loss of a single RB1 allele induces

replication stress and defects in

chromosome segregation.

• RB1 haploinsufficiency results in

aneuploidy that may contribute to

tumor formation.

pRB Regulates Genome Stability in a Dosage-Sensitive Manner

Note: In This Issue is written by Cancer Discovery Science Writers. Readers are encouraged to consult the original articles for full details.



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