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The only potentially curative treatment for myeloproliferative neoplasms (MPN) is hematopoi-etic stem cell transplantation, and this approach is of limited use in this patient population due to factors including lack of donors, patients’ advanced age, and comorbidities. Frameshift mutations affecting the C ter-

minus of the MPN driver calreticulin (CALR) are common in MPN and generate a 36-amino-acid tail dissimilar to that of the wild-type CALR, making it an attractive neoanti-gen target. Cimen Bozkus and colleagues found that some patients with CALR-mutant MPN exhibited T-cell responses—predominantly the CD4+ T-cell phenotype—that were specifi c to mutant CALR. One possible explanation for the fact that the response was seen in only some patients is expression

of programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte antigen 4 (CTLA4); indeed, in vitro experiments showed that blockade of checkpoint receptors restored the immune response to mutant CALR, and in vivo PD-1 block-ade with pembrolizumab promoted a T-cell response in one patient with CALR-mutant MPN. Promisingly, it was possible to stimulate CD4+ and CD8+ T cells from healthy donors with no prior exposure to mutant CALR to specifi cally react to mutant CALR, ignoring wild-type CALR. Additionally, T cells recognize several endogenously processed and presented epitopes in the unique C terminus of mutant CALR. Col-lectively, these fi ndings indicate that, in patients with CALR-mutant MPN, immunotherapies targeting the mutant CALR protein via neoantigen-specifi c vaccines or adoptive T-cell therapies may be worth investigating, as may immune check-point blockade therapies. ■

See article, p. 1192.

• Some patients with myeloprolifera-tive neoplasms (MPN) had T-cell re-sponses to mutant calreticulin (CALR).

• Pembrolizumab promoted this T-cell response, and healthy-donor T cells could react to mutant CALR.

• Patients with CALR-mutant MPN may benefit from T-cell therapies or from immunotherapy targeting mutant CALR.

Mutant Calreticulin Is a Shared Neoantigen in Myeloproliferative Neoplasms

Mutations in RAS/RAF/MEK/ERK pathway members that constitutively activate ERK lead to increased signaling through this pathway, driving tumor growth. Allosteric MEK inhibi-tors have been developed and used in combination with RAF inhibitors to treat BRAFV600 mutant–driven cancers. Resist-

ance mechanisms to RAF and MEK combination therapy have been defi ned; they mostly function by decreasing the pharmacologic activity of RAF inhibitors. The mechanisms that underlie resistance to MEK inhibitors in the clinic remain largely unknown. Gao and colleagues report a patient with colon cancer with a MEK1 mutation (V211D) acquired during treatment with the MEK inhibitor bini-metinib and the anti-EGFR antibody panitumumab. This mutation was not seen in any of more than 30,000 sequenced

clinical specimens and appeared to be treatment-emergent. Unlike wild-type MEK, the phosphorylation of MEK1V211D

was only partially dependent on the upstream RAF kinase and exhibited elevated baseline catalytic activity. MEK1V211D

was not susceptible to allosteric MEK inhibitors in vitro or in a mouse fi broblast line; however, MEK1V211D was sensitive to ATP-competitive inhibitors, which inhibited the activity of wild-type and V211D MEK1 equipotently in vitro and in vivo. Experiments using a patient-derived xenograft (PDX) model revealed that ATP-competitive MEK inhibitors caused tumor regression, inhibited ERK signaling, and induced cleaved caspase-3, a marker of apoptosis. Based on the biochemi-cal characterization of this activating MEK mutation as a gatekeeper mutation for allosteric MEK inhibitors, inves-tigation of selective ATP-competitive inhibitors of MEK or MEK inhibitors with different binding sites is warranted in patients with resistance to allosteric MEK inhibitors. ■


• An acquired mutation in MEK1 was found in a patient with colon cancer treated with MEK and EGFR inhibitors.

• MEK1V211D had increased catalytic activity and decreased binding affinity to allosteric inhibitors.

• MEK1V211D was sensitive to ATP-competitive inhibitors, which caused PDX tumor regression.

A MEK1 Mutant Resists Allosteric but Not ATP-Competitive Inhibitors

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1144 | CANCER DISCOVERY�SEPTEMBER 2019 www.aacrjournals.org

Although more than half of patients with breast cancer are 60 years of age or older at the time of diagnosis, little is known about how age affects treatment responses. Using a mouse triple-negative breast cancer (TNBC) model, Sceneay, Goreczny, and colleagues discovered that agedanimals had diminished res-

ponses to immune checkpoint blockade (ICB) with anti–PD-L1 or anti-CTLA4. ICB therapy caused enrichment of interferon and infl ammatory response pathways in respond-ing tumors from young mice, but not those of aged mice. Increased age correlated with enhanced CD8+ T-cell effector memory and exhaustion phenotypes, and ex vivo stimulation of CD8+ T cells led to enhanced expression of checkpoint pro-teins (PD-1, CTLA4, LAG3, and TIM3) on the cells from aged

mice. Even without treatment, there were marked differences in the immune contexture of the tumor microenvironment between young and aged mice; interferon and infl ammatory responses and antigen presentation and processing genes were reduced in tumors from aged mice. Similarly, in patients with TNBC, those 40 years or younger in age had increased expression of antigen-processing and interferon and infl am-matory response pathway genes relative to those 65 years or older. Intratumoral injection of a mouse-specifi c stimulator of interferon genes agonist, DMXAA, in combination with anti–PD-L1 or anti-CTLA4 led to reduced tumor growth and increased survival in aged mice, whereas addition of DMXAA did not increase effi cacy of ICB in young mice. These results suggest that patients may benefi t from assessment of inter-feron pathway status to determine need for additional immu-notherapy in combination with ICB. ■


• Aged mice exhibited immune dys-function that reduced efficacy of ICB in a TNBC model.

• Addition of a STING agonist increased efficacy of immune checkpoint blockade in aged mice.

• Age and status of interferon-related genes may have implica-tions for management of TNBC.

Age Affects Immune Checkpoint Blockade Efficacy in TNBC

Although epigenetics and meta-bolism both contribute to can-cer pathogenesis, little is known about how epigenetics may infl u-ence metabolism in cancer. Gu, Liu, and colleagues found that mutations in some subunits of polycomb repressive complex 2 (PRC2) and an activating muta-tion in neuroblastoma RAS

(NRASG12D) worked together to cause progression of myelo-proliferative neoplasms (MPN) to primary myelofi brosis and promote leukemic transformation in human hematopoietic cells and mice. However, complete loss of PRC2 function via inactivation of the PRC2 component enhancer of zeste homolog 1 (EZH1) in EZH2-KO NRASG12D+/− mice abol-ished MPN progression. Because EZH1 function is dispen-sable in normal hematopoietic stem cells, EZH1-targeted

therapies may be worth investigating for EZH2-mutant leukemia. RNA-sequencing experiments revealed that the branched-chain amino acid (BCAA) metabolism pathway was upregulated in hematopoietic/stem progenitor cells of EZH2-KO NRASG12D+/− mice relative to NRASG12D+/− mice, with BCAT1—the fi rst enzyme involved in catalyzing BCAA transamination—being specifi cally epigenetically reac-tivated in EZH2-defi cient cells to produce excess BCAAs. NRASG12D cooperated with loss of EZH2 to provide substrate for BCAT1-mediated transamination, and EZH2-defi cient, NRAS-mutant leukemia-initiating cells had increased BCAA pools and were dependent on BCAA-driven mTOR signal-ing for survival. Because BCAT1 loss does not affect normal HSPCs and BCAAs can be restricted, BCAT1 and BCAAs may be useful targets to investigate in hematologic malignancies, particularly those with EZH2 loss and/or NRAS mutation. ■


• EZH2 knockout combined with NRAS activation promoted leukemic transfor-mation of myeloproliferative neoplasms.

• Loss of EZH2 leads to epigenetic reac-tivation of BCAT1, and mutant NRAS promotes BCAT1 transamination.

• EZH2-deficient leukemia-initiating cells have increased BCAA pools and depend on BCAA-driven signaling.

EZH2 Loss and NRAS Activation Cooperate to Promote MPN Progression



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SEPTEMBER 2019�CANCER DISCOVERY | 1145

Glioma stem cells (GSC) con-tribute to pathogenesis and treat-ment ineffi cacy in glioblastoma. To determine molecular pro-cesses and potential vulnerabili-ties unique to GSCs, Gimple and colleagues identifi ed GSC-spe-cifi c superenhancer-associated genes that were associated with poor prognosis in glioblastoma

and had elevated expression in glioblastoma compared with other brain cells. Among such genes, the long-chain polyun-saturated fatty acid (LC-PUFA) synthesis gene ELOVL2 sup-ported GSC proliferation by promoting LC-PUFA synthesis, which GSCs relied on to maintain membrane composition and integrity and to support effi cient EGFR signaling at the cell membrane. Demonstrating the LC-PUFA synthesis pathway’s clinical relevance, high-grade gliomas had more

direct products of ELOVL2 than low-grade gliomas, and lipid-metabolism synthesis gene signatures correlated with poor clinical outcome. Further, FADS2, the enzyme imme-diately downstream of ELOVL2 in LC-PUFA synthesis, was upregulated in glioblastoma and GSCs, and FADS2 deple-tion diminished GSC proliferation. GSCs treated with an EGFR inhibitor (lapatinib) and a FADS2 inhibitor (SC-26196) reduced proliferation more than either drug alone. Addition-ally, in a mouse xenograft model, ELOVL2 knockdown slowed tumor formation, and treatment with lapatinib, SC-26196, or both prolonged survival. Collectively, these results imply there may be a positive feedback loop in which LC-PUFA synthesis supports cell membrane structure, which promotes EGFR signaling. Because therapies that target EGFR alone have failed in glioblastoma, testing a combined approach that also targets LC-PUFA synthesis may be warranted. ■


• Glioma stem cells (GSC) exhibit distinct superenhancer landscapes compared with normal brain tissue.

• ELOVL2 expression enhances GSC proliferation and EGFR signaling by regulating membrane composition.

• Targeting EGFR and ELOVL2-mediated fatty-acid synthesis may be a useful strategy in glioblastoma.

Glioma Stem Cells Depend on Superenhancer-Driven ELOVL2 Upregulation

Activating mutations in KRAS are nearly ubiquitous in pan-creatic ductal adenocarcinoma (PDAC), but oncogenic KRAS has not been effectively targeted. Humpton, Alagesan, and col-leagues discovered that in mouse embryonic fi broblasts and pan-creatic ductal organoids, onco-genic KRAS expression reduced

cytoplasmic and mitochondrial reactive oxygen species (ROS) levels and resulted in a reduced mitochondrial network, as meas-ured by mitochondrial mass and membrane potential per cell as well as quantitation of transmission electron micrographs. Oncogenic KRAS expression also led to increased expression of Bnip3l/Nix and an increase in NIX protein in mitochondria, causing increased selective removal of damaged mitochondria (mitophagy), which supports cancer progression in the context of cytotoxic stress by reducing mitochondrial ROS. Further, in

a mouse PDAC model, NIX deletion delayed cancer progres-sion and restored normal levels of functional mitochondria. In human pancreatic cancer cell lines, NIX depletion decreased mitophagic fl ux and diminished proliferation in glucose-lim-ited conditions—similar to those in PDAC—likely by increas-ing the requirement for NADPH. Notably, however, increased mitochondrial clearance content in NIX-ablated KPC mice was eventually overcome by activating alternative pathways of mito-chondrial clearance, so mitochondrial content was ultimately reduced to normal levels, allowing cancer progression despite a substantial extension of median survival. Demonstrating the potential clinical relevance of these fi ndings, analysis of data from The Cancer Genome Atlas revealed that elevated NIX expression was associated with reduced survival in patients with PDAC. These results imply that therapy targeting mitophagy, possibly in combination with ROS-generating drugs, may be worth investigating for PDAC. ■


• Mutations that activate KRAS led to increased expression of NIX in a mouse model of pancreatic cancer.

• NIX overexpression increased removal of undamaged mitochondria (mito-phagy), promoting cancer progression.

• In glucose-limited conditions, NIX depletion reduced mitophagic flux and proliferation of PDAC cells.

NIX-Mediated Mitophagy Contributes to Pancreatic Cancer Pathogenesis



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1146 | CANCER DISCOVERY�SEPTEMBER 2019 www.aacrjournals.org

Disease outcome in pancre-atic ductal adenocarcinoma (PDA) is affected by the com-position of infl ammatory cells in the tumor microenvironment (TME), and unconventional T-lymphocyte populations are now being recognized as regu-lators of tumor immunity and disease. Hundeyin, Kurz, and

colleagues found that one such T-lymphocyte population, TCRαβ+CD4−CD8−NK1.1− innate αβ T cells (iαβT), com-prised 10% of the infi ltrating T-lymphocytes in a mouse model of PDA and in human PDA tumors. iαβTs in the TME were highly activated and exhibited a unique phenotype characterized by expression of the adhesion ligand JAML and the cytotoxic marker CD107a along with upregulation of the C-type lectin receptor Dectin-1 and several checkpoint

receptors, ectonucleotidases, and costimulatory receptors. iαβT transfer also exerted protective effects against PDA in mice, reducing tumor growth, activating CD4+ and CD8+ T cells in the TME, and increasing expression of CD44, ICOS, and TNFα. Additionally, the TME of iαβT-treated tumors was immunogenically reprogrammed, and CD4+ and CD8+

T-cell populations increased substantially. Consistent with these mouse fi ndings, autologous iαβT treatment in patient-derived organotypic tumor spheroids caused conventional T-cell activation. iαβTs did not reduce proliferation or pro-mote lysis or apoptosis of PDA tumor cells, indicating that they are not directly cytotoxic to the PDA cells. However, iαβTs did indirectly aid the adaptive immune response by activating CCR5, thus promoting immunogenic macrophage polarization. Together, these results imply that cell therapies based on iαβTs may be of interest in PDA. ■


• The unconventional T cells iαβTs infil-trate pancreatic ductal adenocarci-noma tumors and reduce tumor growth.

• iαβTs cause CD4+ and CD8+ activa-tion in mice and conventional T-cell activation in a human model.

• iαβT-based cell therapies may be worth investigating for pancreatic ductal adenocarcinoma.

Immunogenic i`aT Cells Infiltrate Pancreatic Ductal Adenocarcinoma

PTEN, a tumor suppressor often disabled in cancers, is a lipid and protein phosphatase that has a variety of functions, including in repair of DNA double-strand breaks (DSB). In multiple cell lines, Zhang, Lee, Dang, and colleagues found that the presence of DNA DSBs increased PTEN phosphoryla-

tion at T/S398 by ATM kinase, promoting PTEN’s inter-action with mediator of DNA damage checkpoint protein 1 (MDC1), which mediated PTEN’s interaction with the histone-lysine N-methyltransferase NSD2 (also known as MMSET/WHSC1). K349 of PTEN was then dimethylated by NSD2; this dimethylated lysine was subsequently recognized

by the tudor domain of p53-binding protein 1, promot-ing PTEN accumulation on the DNA DSB sites. Moreover, NSD2-mediated K349 dimethylation of PTEN was required for effi cient DNA DSB repair. The potential clinical relevance of this discovery is illustrated by the fact that inhibition of NSD2-mediated PTEN dimethylation made human colorec-tal carcinoma cells and cancer cells in xenografted mice more susceptible to PI3K-inhibitor treatment and DNA damage caused by etoposide or irradiation. This effect appeared to be dependent on PTEN protein phosphatase activity–medi-ated dephosphorylation of the histone protein γH2AX, a biomarker for DNA DSBs. In summary, this work provides insight into the molecular mechanism of PTEN-mediated DNA DSB repair and its possible role in cancer. ■


• DNA double-strand breaks promoted PTEN dimethylation and interaction with histone methyltransferase NSD2.

• Following dimethylation, PTEN was recruited to DNA double-strand break sites by 53BP1.

• Inhibiting PTEN dimethylation in-creased cancer cells’ sensitivity to PI3K inhibitors with DNA-damaging agents.

Recruitment of PTEN to DNA Damage Sites Depends on Dimethylation

In This Issue is written by Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details.



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