Addiction to Multiple Oncogenes Can Be Exploited to Prevent the Emergence of Therapeutic Resistance

Addiction to multiple oncogenes can be exploited to prevent the emergence of therapeutic resistance

Peter S. Choi, Yulin Li, and Dean W. Felsher1

Division of Oncology, Departments of Medicine, Pathology, and Molecular Imaging, Stanford University School of Medicine, Stanford, CA 94305

Edited by Stephen R. Hann, Vanderbilt University, Nashville, TN, and accepted by the Editorial Board July 2, 2014 (received for review April 2, 2014) Many cancers exhibit sensitivity to the inhibition of a single genetic therapy for cancer, it has been proposed that a similar strategy, lesion, a property that has been successfully exploited with using combinations directed against multiple dependencies, is the oncogene-targeted therapeutics. However, inhibition of single most likely to prevent resistance (7). Indeed, mathematical mod- oncogenes often fails to result in sustained tumor regression due eling indicates that targeting at least two independently re- to the emergence of therapy-resistant cells. Here, we report that quired pathways may be sufficient to prevent tumor recurrence MYC-driven lymphomas frequently acquire activating mutations in (15). However, there exists little experimental evidence directly β-catenin, including a previously unreported mutation in a splice testing such an approach and it remains unclear which com- acceptor site. Tumors with these genetic lesions are highly depen- binations of targets would be most effective at inducing long- dent on β-catenin for their survival and the suppression of β-catenin term remissions. resulted in marked apoptosis causally related to a decrease in Bcl-xL MYC is one of the most frequently amplified oncogenes in expression. Using a novel inducible inhibitor of β-catenin, we illus- human cancer (16). In the Eμ-tTA/tetO-MYC conditional mouse trate that, although MYC withdrawal or β-catenin inhibition alone model, overexpression of MYC results in the development of results in initial tumor regression, most tumors ultimately re- aggressive T-cell lymphoma, and MYC inactivation in estab- curred, mimicking the clinical response to single-agent targeted lished tumors is sufficient to induce tumor regression through therapy. Importantly, the simultaneous combined inhibition of processes such as proliferative arrest, cellular senescence, apo- β both MYC and -catenin promoted more rapid tumor regression ptosis, and the shutdown of angiogenesis (17–19). The extent of and successfully prevented tumor recurrence. Hence, we demon- regression is dependent on both cell-intrinsic and host-dependent β strated that MYC-induced tumors are addicted to mutant -catenin, contexts, and in particular, tumors frequently recur following β and the combined inactivation of MYC and -catenin induces sus- MYC inactivation in the absence of an intact adaptive immune tained tumor regression. Our results provide a proof of principle system (20). Recurring tumors restore expression of the MYC that targeting multiple oncogene addicted pathways can prevent transgene or up-regulate expression of endogenous Myc,dem- therapeutic resistance. onstrating that resistance occurs primarily through reactivation of the MYC pathway (21). Thus, MYC oncogene addiction and oncogene addiction | combination targeted therapy | splice site mutations tumor recurrence in the Eμ-tTA/tetO-MYC lymphoma model resembles the clinical course of human cancers treated with single ancer cells are highly sensitive to the targeted inhibition of agent targeted therapy. Csingle driver mutations, eliciting a phenomenon known as Here, we demonstrate that the combined inactivation of two “ ” oncogene addiction (1). The identification of genetic dependencies oncogene addiction pathways can result in sustained tumor re- in multiple tumor types has resulted in the development of sev- gression. Moreover, we describe a previously unidentified splice eral molecularly targeted therapeutics, including the BCR-ABL acceptor site mutation in β-catenin that is associated with MYC- kinase inhibitor imatinib for the treatment of chronic myeloge- induced lymphomagenesis. Tumors with mutations in β-catenin nous leukemia (CML), the EGFR kinase inhibitor gefitinib for – the treatment of non small cell lung cancer (NSCLC), and the Significance BRAF kinase inhibitor vemurafenib for the treatment of advanced melanoma (2–4). Although these oncogene-targeted agents have provided promising clinical responses, many patients ultimately The rationale of targeting specific genetic dependencies for the experience a recurrence of their disease due to the development of treatment of cancer has been validated by the promising clin- drug resistance (4–6). Thus, it has become evident that mono- ical responses obtained with oncogene-targeted therapies. therapy with targeted drugs is insufficient for achieving sustained However, in most cases, the development of resistance remains a major obstacle toward achieving long-term or complete dis- tumor regression. ease remission. Here, we identified mutant β-catenin as a com- Resistance to targeted therapy can arise through multiple mech- mon secondary oncogene addiction pathway in MYC-addicted anisms, depending on the tumor type and the targeted oncogenic lymphoma. We demonstrate that, although withdrawal or pathway (7). Cells frequently acquire resistance through mutations inhibition of either oncogene is sufficient to induce initial in the targeted oncogene itself that disrupt drug binding, as in the tumor regression, only combined inhibition of both oncogene case of BCR-ABL and EGFR (8, 5, 6). In addition, resistance to addiction pathways results in sustained tumor regression. Our EGFR inhibition in NSCLC and BRAF inhibition in melanoma results suggest clinical outcomes can be dramatically improved has been found to occur through a variety of mechanisms that through the simultaneous targeted inhibition of multiple onco- activate downstream signaling proteins or alternative pathways, genic addiction pathways. which can functionally substitute for loss in activity of the targeted – oncogene (9 11). Although significant progress has been made Author contributions: P.S.C., Y.L., and D.W.F. designed research; P.S.C. and Y.L. performed in the identification and inhibition of resistance pathways, it may research; P.S.C. and Y.L. analyzed data; and P.S.C. and D.W.F. wrote the paper. prove challenging to anticipate and suppress all of the potential The authors declare no conflict of interest. mechanisms of resistance for each oncogene-addicted cancer and This article is a PNAS Direct Submission. S.R.H. is a guest editor invited by the Editorial targeted therapeutic agent. Board. Combination therapy has been successfully applied to prevent 1To whom correspondence should be addressed. Email: [email protected]. resistance in the treatment of infectious diseases such as HIV This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (12, 13) and tuberculosis (14). In the context of oncogene-targeted 1073/pnas.1406123111/-/DCSupplemental.

E3316–E3324 | PNAS | Published online July 28, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1406123111 Downloaded by guest on September 27, 2021 PNAS PLUS

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Fig. 1. MYC-induced lymphomas frequently harbor stabilizing mutations in β-catenin. (A)InEμ-tTA/tetO-MYC transgenic mice, T-cell lymphomas are proposed to arise as a result of MYC overexpression and alterations in unidentified cooperating pathways. (B) Distribution of β-catenin mutations detected by sequencing of DNA from Eμ-tTA/tetO-MYC primary lymphoma specimens. (C) Western blot analysis for β-catenin protein in primary MYC-induced lymphomas and lymphoma-derived cell lines. β-actin is shown as a loading control. Closed and open triangles denote full-length and Δexon3 β-catenin protein, re- spectively. Tumors with mutant β-catenin are labeled in red. (D) Schematic of β-catenin depicting the phosphorylation residues encoded by exon 3 that are critical for protein degradation. Also shown are sequence chromatograms of mutations in phosphorylation residues identified in primary MYC-induced lymphomas. (E) Diagram of conserved splice donor and acceptor sites required for proper splicing of the β-catenin primary transcript. Also shown are sequence chromatograms of splice acceptor site mutations identified in primary MYC-induced lymphomas. (F) Relative expression levels of transgenic MYC and the β-catenin/TCF target genes Axin2 and Enc1 across a panel of MYC-induced primary lymphomas with WT (n = 8) or mutant (n = 6) β-catenin, as analyzed by qRT-PCR. Shown are means ± SEM with **P < 0.01.

Choi et al. PNAS | Published online July 28, 2014 | E3317 Downloaded by guest on September 27, 2021 are also highly addicted to this mutant gene product for their discovered novel mutations at the exon 3 splice acceptor site that survival. We demonstrate that in MYC-induced lymphomas, com- are predicted to result in skipping of exon 3 during processing of bined addiction to both MYC and β-catenin can be exploited in a the primary transcript and an in-frame deletion of the region rational manner to prevent the emergence of therapeutic resistance. required for β-catenin degradation (Fig. 1E). Skipping of exon 3 was confirmed at the mRNA level by sequencing of β-catenin Results cDNA from lymphoma samples with splice site mutations (Fig. MYC-Induced Lymphomas Frequently Acquire Stabilizing Mutations in S1). In total, we detected β-catenin mutations in ∼37% of pri- β-Catenin. Lymphomas initiated by MYC overexpression often mary lymphomas from Eμ-tTA/tetO-MYC mice (11 of 30; Fig. acquire loss-of-function mutations in tumor suppressor genes 1B and Table S1). The type of mutation correlated with the size such as p53 and p19ARF (22). However, it remains less well of β-catenin protein that was detected, with elevated levels of known whether MYC overexpression also selects for the acti- full-length protein in samples with exon 3 missense mutations vation of additional oncogenic drivers during lymphomagenesis and expression of a shorter-length protein in samples with splice (Fig. 1A). We discovered that a subset of MYC-induced lym- site mutations (Fig. 1C). Mutations in β-catenin also correlated phomas and lymphoma-derived cell lines from the Eμ-tTA/tetO- with significant up-regulation of the β-catenin/T-cell factor MYC model expressed elevated levels of β-catenin, with both (TCF) target genes Axin2 (ninefold higher in mutant vs. WT, P < full-length and shorter forms of β-catenin protein detectable by 0.01) and Enc1 (21-fold higher in mutant vs. WT, P < 0.001; Western blot (Fig. 1C). To determine the mechanism of β-catenin Fig. 1F) (23, 24). Taken together, our results demonstrate that up-regulation in our panel of MYC-induced lymphomas, we mutations leading to stabilization of β-catenin occur frequently isolated genomic DNA and sequenced the entire β-catenin exon during MYC-induced lymphomagenesis. 3 region and portions of the upstream and downstream introns. We detected missense mutations in 7 of 30 samples, at or ad- MYC-Induced Lymphomas Exhibit Addiction to Mutant β-Catenin. jacent to the phosphorylation sites required for β-catenin deg- Due to the frequency of stabilizing β-catenin mutations in MYC- radation (Fig. 1D). Interestingly, in 4 of 30 samples, we also induced lymphomas, we speculated that β-catenin might be

A B shControl shβcat-2 1.2 cat-2 cat-3 β shRNA: shControl shβ sh shβcat-4 1.0 full-length β-catenin 0.8 Δexon3 0.6

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Fig. 2. MYC-induced lymphomas exhibit addiction to mutant β-catenin. (A) Western blot analysis of MYC lymphoma cells retrovirally infected with either control shRNA (shControl) or three independent shRNAs targeting β-catenin (shßcat-2, shßcat-3, and shßcat-4). (B) Relative mRNA expression levels of transgenic MYC, β-catenin, and three β-catenin target genes (Axin2, Runx2, and Enc1) in MYC lymphoma cells infected with either control or β-catenin shRNA, as analyzed by qRT-PCR. (C) Representative histograms and (D) quantification of GFP fluorescence in MYC lymphoma cells after infection with either control or β-catenin shRNA coexpressing GFP. (E and F) Quantification of GFP fluorescence over time of MYC lymphoma cell lines mutant or WT for β-catenin and infected with retrovirus expressing either (E) β-catenin shRNA or (F)theβ-catenin inhibitor ICAT. Values represent means ± SD of two cell lines with WT + β-catenin and two cell lines with mutant β-catenin and three independent experiments per cell line. Data are normalized to the percentage of GFP cells at day 1 after infection.

E3318 | www.pnas.org/cgi/doi/10.1073/pnas.1406123111 Choi et al. Downloaded by guest on September 27, 2021 required for tumor maintenance. We used a retroviral vector A PNAS PLUS expressing β-catenin–specific shRNAs and a GFP reporter to pMSCV-DD-ICAT TCF infect MYC lymphoma cell lines known to be mutant or WT for LTR DD ICAT Target genes Destabilizing β-catenin βcat β-catenin. Efficient knockdown was observed with three dif- domain inhibitor ferent shRNAs targeting β-catenin (Fig. 2A), and suppression + βcat of β-catenin levels resulted in significant depletion of GFP −TMP +TMP ICAT cells over time compared with a control nontargeting shRNA DD ICAT DD ICAT DD (Fig. 2 C and D). Sensitivity to β-catenin knockdown was also DD-ICAT DD-ICAT β-catenin observed for an additional 13 MYC lymphoma lines that were degraded stable off β A − − characterized as mutant for -catenin (Fig. S2 ). In contrast, B Time of TMP treatment (hours) C DOX: + + TMP: − − + + MYC lymphoma lines identified as WT for β-catenin were 0 0.5 1 2 3 48 completely resistant to β-catenin suppression (Fig. 2E), con- MYC DD-ICAT firming a strong correlation between β-catenin mutation status DD-ICAT and dependency. Therefore, MYC-induced lymphomas are α-tubulin highly dependent on continued expression of mutant β-catenin. ICAT (also known as CTNNBIP1) is an endogenously ex- α-tubulin pressed gene that was first identified in a screen of β-catenin interacting proteins and found to inhibit the interaction between D β-catenin/TCF target genes E 7xTCF luciferase reporter 1.2 1.2

β ty -catenin and its transcriptional partner TCF (25). Similar to – TMP i – TMP β-catenin knockdown by shRNA, overexpression of ICAT also 1.0 +TMP 1.0 +TMP resulted in specific depletion of MYC lymphoma cells expressing 0.8 0.8 xpression mutant, but not WT, β-catenin (Fig. 2F). In contrast, expression 0.6 0.6 of dominant-negative TCF4 (TCF4ΔN31) was growth suppres- 0.4 0.4 sive to both β-catenin WT and mutant MYC lymphomas (Fig. Relative0.2 e 0.2 Relative luciferase activ B β 0.0 (Firefly/Renilla luciferase) 0.0 S2 ). Several reported chemical inhibitors of -catenin also did Axin2 Enc1 Mecom Runx2 Control DD-ICAT not exhibit selective activity when used to treat either β-catenin WT or mutant MYC lymphomas (Fig. S2C), demonstrating that Fig. 3. Inducible inhibition of β-catenin through a chemical-genetic ap- β these compounds have significant off-target effects in MYC proach. (A) Schematic outlining the inducible DD-ICAT -catenin inhibitor consisting of a degradation domain (DD) and the β-catenin inhibitor ICAT. lymphoma cells. Collectively, these results indicate that MYC β DD-ICAT is constitutively degraded and becomes stabilized in the presence lymphomas are sensitive to inhibition of mutant -catenin, either of the ligand TMP. Once stabilized, DD-ICAT prevents the interaction be- through shRNA-mediated knockdown or by expression of the tween β-catenin and TCF and effectively inhibits β-catenin transcriptional inhibitory protein ICAT. activity. (B) Western blot analysis of MYC lymphoma cells expressing DD- ICAT and treated with TMP for 0–8h.(C) Western blot analysis of MYC Inducible Inhibition of β-Catenin Through a Chemical-Genetic Approach. lymphoma cells expressing DD-ICAT treated with either DOX alone, TMP In lymphoma cells from the Eμ-tTA/tetO-MYC model, expression alone, or both DOX and TMP. (D) Relative expression of β-catenin target of the MYC transgene is driven by the tetracycline-regulatory genes Axin2, Enc1, Mecom/Evi1,andRunx2 in MYC lymphoma cells system (tet-system) and is suppressed in the presence of tetracy- expressing DD-ICAT and treated with either DMSO or TMP for 2 d. (E) Quantification of luciferase signals in MYC lymphoma cells infected with cline or tetracycline analogs such as doxycycline (DOX). To sup- × β a7 TCF binding site luciferase reporter and DD-ICAT, treated with either press -catenin activity in a similarly inducible manner, we took DMSO or TMP for 24 h. Values are means ± SD of normalized firefly/renilla advantage of a recently developed expression system in which luciferase ratios from three independent experiments. protein stability can be controlled by the presence or absence of a specific chemical ligand (26). A gene of interest can be fused in- frame to a mutant destabilized form of Escherichia coli dihy- temporally controlled and effectively suppresses β-catenin– drofolate reductase (ecDHFR), resulting in constitutive degrada- dependent transcription. tion of the entire protein product. Addition of the DHFR ligand trimethoprim (TMP) leads to rapid stabilization of the fusion Inhibition of β-Catenin Induces Apoptosis Through Regulation of Bim protein (Fig. 3A). TMP is highly selective for bacterial DHFR and and Bcl-xL Expression. We next sought to take advantage of the has favorable pharmacological properties, making the ecDHFR- DD-ICAT inducible system to characterize the dependence of TMP conditional expression system well suited for both in vitro and MYC-induced lymphomas on the β-catenin pathway. MYC in vivo studies in mammalian cells. To generate an inducible in- lymphoma cells expressing DD-ICAT were treated with TMP, β hibitor of -catenin, we fused the destabilizing ecDHFR domain and levels of cell death over time were measured by staining for MEDICAL SCIENCES (DD) to the N terminus of ICAT (Fig. 3A) and stably expressed the the cell surface apoptotic marker Annexin-V and the DNA dye DD-ICAT fusion in a MYC lymphoma cell line. Levels of DD- 7-aminoactinomycin D (7-AAD). Inhibition of β-catenin activity ICAT were low in the absence of TMP and were rapidly induced via induced expression of DD-ICAT resulted in an increase in B with addition of TMP (Fig. 3 ). the total percentage of apoptotic cells, from a basal level of + + We first confirmed orthogonal regulation of MYC and DD- ∼13% Annexin-V cells before treatment to 64% Annexin-V ICAT by treating cells with DOX alone, TMP alone, or a com- cells after 3 d of treatment with TMP (Fig. 4A). In contrast to bination of DOX and TMP. MYC levels were only suppressed in MYC inactivation, which induces both cell cycle arrest and ap- the presence of DOX, whereas DD-ICAT expression was only β induced in the presence of TMP (Fig. 3C). Next, we investigated optosis (17), we found that -catenin inhibition had no signifi- the effects of DD-ICAT induction on β-catenin activity. Treat- cant effect on the percentage of proliferating cells, as determined ment with TMP and stabilization of DD-ICAT resulted in down- by staining for DNA content with propidium iodide (PI) (Fig. E regulation of the β-catenin/TCF target genes Axin2, Enc1, Mecom, 4 ). As a control, TMP-induced expression of a DD-YFP fusion and Runx2 (Fig. 3D) (23, 24, 27, 28), as well as down-regulation of protein had no effect on the survival of MYC lymphoma cells a luciferase reporter driven by a 7× repeat of the canonical TCF both in vitro and in vivo (Fig. S3 A–D), demonstrating the lack of binding sequence (Fig. 3E) (29). Our results demonstrate the any nonspecific toxicity due to expression of the ecDHFR activity of a novel inducible inhibitor of β-catenin that can be destabilizing domain or treatment with TMP.

Choi et al. PNAS | Published online July 28, 2014 | E3319 Downloaded by guest on September 27, 2021 A β-catenin on β-catenin off 1d β-catenin off 2d β-catenin off 3d 7.4% 10.3% 29.1% 46.7% 7-AAD

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Fig. 4. Mutant β-catenin is required for survival of MYC lymphomas through regulation of apoptotic factors. (A) MYC lymphoma cells expressing DD-ICAT were treated with TMP and levels of apoptosis were tracked by staining for Annexin-V-PE and 7AAD. (B) Western blot analysis for apoptotic proteins in cells treated with TMP for 0, 1, 2, and 3 d. (C) Levels of proapoptotic Bim and antiapoptotic Bcl-xL mRNA in cells treated with TMP for 0, 1, 2, and 3 d, as determined by qRT-PCR. (D) Quantification of apoptosis as measured by staining with Annexin-V after treatment with TMP in cells expressing Bim shRNA or Bcl-xL cDNA compared with empty vector control. All values represent means of at least three independent experiments ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (E) MYC lymphoma cells expressing DD-ICAT were either untreated, treated with TMP (1 μM), or treated with DOX for 24 h. Cells were fixed and stained with PI to measure DNA content. Histogram events are gated on viable cells.

To further characterize the mechanism of apoptosis due to hibition of a single oncogene induced significant short-term re- inhibition of β-catenin, we collected cells that were treated with gression, all tumors eventually recurred, with resistant tumor TMP for 0, 1, 2, and 3 d and screened the expression of several cells emerging after ∼2 wk of continuous treatment. Strikingly, pro- and antiapoptotic factors by Western blot (Fig. 4B). In- the combined inhibition of both MYC and β-catenin resulted in hibition of β-catenin led to an increase in levels of cleaved cas- more rapid tumor regression and long-term recurrence-free sur- pase-3, which correlated with an induction in levels of Bim and vival, despite cessation of treatment after only 16 d (Fig. 5 A–D). a reduction in levels of Bcl-xL at both the mRNA and protein Control DD-YFP–expressing tumors developed resistance to levels (Fig. 4 B and C). We observed no changes in the levels of combination treatment with DOX+TMP (Fig. S3E), confirming Birc5, an antiapoptotic factor shown to be a transcriptional the absence of nonspecific toxicity due to TMP treatment or ex- target of β-catenin/TCF in colon carcinoma cells (30) (Fig. 4B). pression of the DD domain. Our findings demonstrate that the In addition, shRNA-mediated knockdown of Bim or ectopic outgrowth of resistant cells can be effectively suppressed by the expression of Bcl-xL was sufficient to significantly dampen the combined inhibition of both MYC and β-catenin. apoptotic response to β-catenin inhibition (Fig. 4D). These To determine the mechanism of enhanced tumor regression results demonstrate that β-catenin is required for the survival of we observed with combined targeting of MYC and β-catenin, we MYC-induced lymphomas by promoting the expression of Bcl-xL stained tumors treated with DOX alone, TMP alone, or DOX+ and suppressing the expression of Bim. TMP, for the proliferation marker Ki-67 and the apoptosis marker cleaved caspase-3 (Fig. 6). Inactivation of MYC or combined in- Combined Inactivation of MYC and β-Catenin Synergizes in Tumor activation of both MYC and β-catenin for 4 d resulted in a greater + Regression and Is Sufficient to Prevent Tumor Recurrence. As we reduction in Ki-67 cells compared with inhibition of β-catenin + have previously shown, MYC-induced lymphomas fail to un- alone (Ki-67 cells: MYC off = 2.0% and MYC+β-cat off = 0.7% dergo sustained tumor regression upon MYC inactivation in the vs. β-cat off = 32.8%, P < 0.001; Fig. 6B). Inhibition of β-catenin absence of an intact host immune system (20). We speculated or combined inactivation of both MYC and β-catenin resulted in that targeting a secondary addiction pathway in addition to MYC more apoptosis after 2 d of treatment compared with withdrawal + might be sufficient to prevent tumor recurrence. To test this of MYC alone (cleaved caspase-3 cells: β-cat off = 10.1% and hypothesis, a cohort of SCID mice was transplanted s.c. with MYC+β-cat off = 8.8% vs. MYC off = 1.7%, P < 0.01; Fig. 6C). MYC-induced lymphoma cells expressing DD-ICAT and a firefly Thus, the simultaneous inactivation of MYC and β-catenin pro- luciferase reporter (LUC2). Tumors were allowed to establish, motes a greater decrease in proliferation and increase in apoptosis and mice were treated with either DOX alone, TMP alone, or compared with inhibition of either oncogene alone. Taken to- a combination of DOX and TMP. Tumor burden was monitored gether, our results demonstrate that combined inhibition of MYC over time by bioluminescence imaging (Fig. 5A). Although in- and β-catenin in MYC-induced lymphomas synergizes to promote

E3320 | www.pnas.org/cgi/doi/10.1073/pnas.1406123111 Choi et al. Downloaded by guest on September 27, 2021 PNAS PLUS A 10 12 C ) ) %

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Fig. 5. Combined targeting of MYC and β-catenin prevents tumor recurrence. (A) MYC lymphoma cells expressing LUC2 and DD-ICAT were transplanted s.c. into SCID recipients. Bioluminescence signal was quantified over time after initiation of treatment to inactivate MYC (+DOX), β-catenin (+TMP), or both MYC and β-catenin (+DOX/TMP). Values are means ± SEM (DD-ICAT +DOX, n = 7; DD-ICAT +TMP, n = 3; DD-ICAT +DOX/TMP, n = 5). (B) Representative bioluminescence images of tumor-bearing mice at 0, 8, and 24 (+TMP, +DOX/TMP) or 26 (+DOX) d after initiation of treatment. (C) Recurrence-free survival of mice transplanted with MYC lymphomas and treated with DOX, TMP, or both DOX and TMP as in A. Tumors were scored as recurrences on the day when the bioluminescence signal increased after initial tumor regression. Survival curves were compared by the log-rank test. (D) Quantification of minimum residual disease in transplanted mice treated with DOX, TMP, or both DOX and TMP. Values are means ± 95% CIs. *P < 0.05, **P < 0.01, ***P < 0.001.

tumor regression and is sufficient to prevent the development required oncogenic dependencies is sufficient to prevent the out- of resistance. growth of resistant cells. β-catenin is tightly regulated at the posttranslational level Discussion through phosphorylation of residues in exon 3 (Fig. 1D), leading The emergence of therapeutic resistance represents a significant to ubiquitination and proteasome-mediated degradation (31). obstacle toward achieving long-term remissions with oncogene- Mutations that abrogate β-catenin phosphorylation and result in targeted therapy. Although rational combinations of targeted protein stabilization have been found in a variety of human agents offer the greatest potential for preventing tumor re- cancers (32). We discovered a previously unidentified mecha- currence, it remains unclear which specific combinations would nism of β-catenin stabilization through point mutations in the provide the greatest therapeutic benefit. Here, we took advan- exon 3 splice acceptor site. Although splice site mutations have tage of a transgenic mouse model system in which tumors fre- been associated with loss of function in several tumor suppressor quently recur after MYC inactivation to investigate the efficacy genes, such as TP53, RB1, and BRCA1 (33–36), there have been MEDICAL SCIENCES of simultaneously inhibiting two driver oncogenic events. We relatively few reports of splicing mutations resulting in the gain discovered that MYC-induced lymphomas frequently acquire of function of an oncogene. Examples to date include deletions activating mutations in a splice acceptor site of β-catenin resulting at the intron 10–exon 11 boundary of the receptor tyrosine ki- in its stabilization. In turn, this mutation correlated with marked nase c-KIT that result in loss of an auto-inhibitory region and sensitivity of tumors to β-catenin inhibition. By generating a novel constitutive activation of the protein (37, 38), and splice acceptor inducible inhibitor of β-catenin, we were able to study the in vivo site mutations in c-MET that lead to skipping of exon 14 and consequences of targeting either MYC or β-catenin independently deletion of a juxtamembrane domain important for negative vs. combined inhibition of both oncogenes. The inhibition of ei- regulation (39). Intragenic deletions in β-catenin that remove all ther oncogene alone uniformly gives rise to resistance, whereas or part of exon 3 have been reported in colorectal carcinoma targeting two oncogenes results in more rapid and sustained tumor (40). We conclude that the β-catenin oncogene can also become regression. Our results specifically identify β-catenin activation activated through single nucleotide substitutions occurring at the through a mutation in a splice acceptor site as an additional driver exon 3 splice acceptor site. event associated with MYC-induced lymphomagenesis. Further, Overexpression of MYC due to genomic amplification, trans- we illustrate that cancers can exhibit addiction to multiple onco- location, or via activation of upstream regulators represents one genic drivers and that the combined targeting of two independently of the most common features in human cancer. In addition, the

Choi et al. PNAS | Published online July 28, 2014 | E3321 Downloaded by guest on September 27, 2021 A MYC off β-catenin off MYC and β-catenin off Untreated 2 days 4 days 2 days 4 days 2 days 4 days

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6 40 6 4 leaved caspase-3+ %Ki

20 C 2 % 0 Days of 0 0 2 4 24 24 Days of 0 24 24 24 treatment: treatment: MYC off β-cat off MYC and MYC off β-cat off MYC and β-cat off β-cat off

Fig. 6. Combined inactivation of MYC and β-catenin synergizes to inhibit proliferation and promote apoptosis in vivo. (A) MYC lymphoma cells expressing DD-ICAT were transplanted s.c. into recipient SCID mice. Mice were either left untreated or treated with DOX (MYC off), TMP (β-catenin off), or both DOX and TMP (MYC and β-catenin off) for 2–4 d. Tumors were harvested and stained for the proliferation marker Ki-67 and the apoptotic marker cleaved caspase-3. Nuclei were stained using DAPI. Representative images are shown. (B and C) Quantification of Ki-67 and cleaved caspase-3 staining, in two to three independent tumors from each time point and five fields per tumor. Values represent means ± SEM. **P < 0.01, ***P < 0.001.

WNT/β-catenin pathway is also frequently activated in cancer, from the inhibition of both MYC and β-catenin (Fig. 5A) com- due to mutations in β-catenin or loss-of-function mutations in pared with inhibition of either oncogene alone. The targeting of negative regulators such as APC and AXIN2 (32). Interestingly, each oncogene elicited distinct cellular outcomes, with MYC in- although MYC is a prominent downstream target of WNT/ activation resulting primarily in cell cycle arrest and β-catenin β-catenin signaling (41), our findings suggest a strong selective inhibition leading mainly to the induction of apoptosis. The ac- pressure for β-catenin functions independent of MYC in our celerated tumor regression upon inactivation of both oncogenes lymphoma model. Wnt/β-catenin pathway activation has also been can then be attributed to the collective loss of both proliferation observed in models of MYC-driven liver cancer, suggesting MYC and survival signals. Taken together, these results indicate that and β-catenin can functionally cooperate during tumorigenesis in MYC and β-catenin appear to play complementary roles to pro- diverse tissue contexts (42, 43). It has also been reported that mote and maintain tumorigenesis. lymphomas initiated by activated β-catenin frequently exhibit The effectiveness of combination therapy in our model system overexpression of MYC due to genomic amplification or rear- may be explained by the low probability of tumor cells with cross- rangement and that MYC is required for β-catenin driven lym- resistance to inhibition of both oncogenes. A similar effect is phomagenesis (44–46), further supporting a cooperative model for believed to be responsible for the success of other types of MYC and β-catenin function during tumorigenesis. combination therapy, including highly active antiretroviral ther- We found that β-catenin inhibition resulted in the induction of apy (HAART) for HIV infection, in which the use of a mixture apoptosis, which closely correlated with decreased levels of the of inhibitors targeting different viral enzymes is effective at antiapoptotic factor Bcl-xL (Fig. 4 A–C). Ectopic expression of preventing the emergence of virions that are resistant to any Bcl-XL was also sufficient to completely abrogate the cell death single drug (12, 13). We conclude that the suppression of re- associated with loss of β-catenin activity (Fig. 4D). β-catenin has sistance with combination targeted therapy will be most effective been shown to directly activate the expression of Bcl-xL (45, 47, by selecting targets that are each essential for tumor mainte- 48), and we found that Bcl-xL is a critical downstream mediator nance but are sufficiently independent as to minimize the like- of the addiction to β-catenin in MYC-induced lymphomas. The lihood of cross-resistance arising from a single mutation. phenotypic consequences of β-catenin inhibition may be explained We provided a proof of concept that the simultaneous tar- by an “oncogenic shock” model, in which the rapid loss of survival geted inhibition of two mutually required oncogenes may be cues such as Bcl-xL and the persistence of proapoptotic signaling sufficient to prevent the emergence of therapeutic resistance. results in an imbalance that favors cell death (49). Interestingly, Additional factors, such as tumor burden and heterogeneity, we observed a synergistic effect on the rate of tumor regression overall tumor mutation rate, and tumor-host interactions, will

E3322 | www.pnas.org/cgi/doi/10.1073/pnas.1406123111 Choi et al. Downloaded by guest on September 27, 2021 likely play a critical role in determining the number and types of from Karl Sylvester, Stanford University, Stanford, CA) and ligated into BglII PNAS PLUS targeted therapies that will be most effective in combination. In and XhoI sites. addition, the ability to target many known and newly discovered β driver events will largely depend on the development of thera- Inducible -Catenin Inhibitor. The N-terminal ecDHFR-DD-ICAT fusion was first peutics against novel classes of drug targets, such as transcription generated using splicing by overlap extension (SOE) PCR. The fused PCR product was then ligated into pMSCV-puro (BglII/XhoI). The N-terminal factors and chromatin regulators. However, as we continue to ecDHFR-DD-YFP fusion was PCR amplified directly from pBMN-DHFR-(R12Y uncover the full landscape of genetic alterations in cancer, the G67S Y100I)-YFP-HA-IRES-HcRed-tandem (obtained from Tom Wandless, identification and combined targeting of multiple oncogene ad- Stanford University, Stanford, CA) and ligated into pMSCV-puro (BglII/XhoI). diction pathways offers great potential toward preventing re- Stabilization of fusion proteins was induced by treatment with 1 μMof sistance and improving therapeutic outcomes. TMP (Sigma).

Materials and Methods TCF Luciferase Reporter Assay. The 7TFP lentiviral Wnt reporter (29) was Transgenic Mice and Lymphoma Cell Lines. All animal experiments were ap- obtained from Addgene (plasmid 24308) and modified to replace the pu- proved by the Institutional Animal Care and Use Committee at Stanford romycin resistance gene with the neomycin resistance gene, generating University. MYC lymphomas and lymphoma-derived cell lines were named for 7TFN. MYC lymphoma cells expressing renilla luciferase alone or both renilla the identifying number of the mouse from which they were obtained. Cell luciferase and DD-ICAT were stably transduced with the 7TFN reporter. The lines were grown in RPMI 1640 media supplemented with 10% (vol/vol) FBS, Dual-Glo Luciferase assay system (Promega) was used to quantify the relative penicillin/streptomycin, and 50 μM β-mercaptoethanol. MYC was inactivated levels of luciferase activity ± 1 μM TMP. Wnt-responsive firefly luciferase by treatment with 20 ng/mL of doxycycline. values were normalized to control renilla luciferase values.

Sequencing of β-Catenin. Genomic DNA was extracted from primary tumors or Tumor Transplantation and Bioluminescence Imaging. Lymphoma cells were cell lines using the Qiagen DNeasy kit or by standard phenol-chloroform transplanted s.c. into the flank of immunodeficient SCID mice. Tumors were purification. A region spanning intron 2 and exon 3 of β-catenin was PCR monitored by caliper measurement and bioluminescence imaging. Treatment + amplified (see primers in Table S2) and directly sequenced (Sequetech). of mice with DOX, TMP, or DOX TMP was initiated when tumors reached Mutant peaks were detected by manual inspection of sequencing chroma- similar bioluminescent signals. Bioluminescent signal from tumors was μ tograms and with Mutation Surveyor (Softgenetics). To sequence β-catenin measured by i.p. injection of mice with 100 L of a solution of D-Luciferin (30 cDNA, RNA was first isolated from cells using the Nucleospin RNA II kit mg/mL) and imaging with the IVIS Spectrum (Perkin-Elmer). Images were (Machery-Nagel), and first-strand cDNA synthesis was carried out with Su- collected and analyzed using Living Image software (Perkin-Elmer). To in- μ perScript II (Invitrogen) using oligo-dT primers. The 5′ region of the β-catenin activate MYC, mice were injected once i.p. with 10 g of DOX in PBS and μ transcript was amplified by PCR (see primers in Table S2) and directly maintained on drinking water containing DOX (100 g/mL), which was sequenced. refreshed weekly. To induce DD-ICAT or DD-YFP, mice were treated by daily i.p. injection of 5 mg of pharmaceutical-grade trimethoprim lactate (ABT- Proliferation and Apoptosis Assays. MYC lymphoma cells were infected with 590; Alpha Diagnostic International) and maintained on drinking water + retrovirus, and the percentage of GFP infected cells was tracked over time containing a suspension of 1 mg/mL of TMP (Trimethoprim-Sulfamethox- azole suspension; Qualitest Pharmaceuticals), which was refreshed every 3 d. using a FACScan flow cytometer. Apoptosis was quantified by staining with Annexin-V-PE and 7-AAD (BD Biosciences). To assess cell cycle status, cells were first fixed in 70% (vol/vol) ethanol in PBS at −20 °C for at least 12 h. Immunofluorescence. Tumor-bearing mice were killed at 0, 2, and 4 d after After washing in PBS, cells were resuspended in PI staining solution (PBS/ MYC inactivation, and transplanted tumors were harvested and fixed in – 0.1% TritonX-100, 0.2 mg/mL RNase A, and 20 μg/mL PI) and incubated for formalin. Paraffin-embedded sections were immunostained with anti Ki-67 30 min at room temperature while protected from light. DNA content was (BD Biosciences, 556003) or anti-cleaved caspase-3 (Cell Signaling Techology, then measured using a FACScan flow cytometer. All flow cytometric data 9661). All antibodies are listed in Table S4. Images were obtained on a Nikon were analyzed with Flowjo software (Tree Star Inc.). microscope and analyzed using CellProfiler software (www.cellprofiler.org/).

Plasmids. For β-catenin shRNA-mediated knockdown, oligos were PCR am- ACKNOWLEDGMENTS. We thank members of the D.W.F. laboratory for helpful suggestions and guidance, Tom Wandless (Stanford University) for plified and cloned into the XhoI/EcoRI sites of the retroviral LMP vector the ecDHFR degradation domain constructs, and Karl Sylvester (Stanford (RNAi Codex/Openbiosystems). Sequences are listed in Table S3. To generate University) for the dominant-negative TCF construct. This work was sup- pMSCV-ICAT-PIG, ICAT was PCR amplified from murine brain cDNA and li- ported by National Institutes of Health (NIH) Training Grant 5 T32 AI07290 (to gated into BglII and XhoI sites. To create pMSCV-TCFΔN31-PIG, dominant- P.S.C.), NIH Grants R01CA170378 and U54CA149145 (to D.W.F.), and Leukemia negative TCF was PCR amplified from pPGS-CITEneo-TCFΔN31 (obtained and Lymphoma Society Translational Research Grant R6223-07 (to D.W.F.).

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