MEK162

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

Gastrointestinal stromal tumors (GISTs) predominantly harbor acti- vating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutantKitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lack-ing the SRC family kinase-binding site on KIT (pY567) exhibited atten-uated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), thecells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/ mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor pro- liferation. The addition of the MEK inhibitor PD-325901 or binime-tinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.GIST | Kit | PI3K | mouseargeted therapies have revolutionized cancer care, but the rapidity with which resistance to single-agent treatments de- velops has unsettled the field. Often the mechanism of resistance is further mutation of the originally targeted oncogene, as occurs with ABL1T315I, KITT670I, EGFRT790M, and ALKL1196M.

Thus,key protumorigenic signals must be emanating from these “on- cogenic drivers.” To date, however, the contribution of individ- ual signal transduction sites on oncogenic drivers has not beendissected using endogenously encoded oncogenes in fully im- munocompetent, genetically engineered models of cancer.Gastrointestinal stromal tumors (GISTs) derive from mesenchy- mal pacemaker cells of the gastrointestinal tract known as interstitial cells of Cajal (ICCs) or their progenitors (1, 2). The receptor tyro- sine kinase KIT is essential for the development of ICCs, and KIT- activating mutations are detected in the majority of GISTs (1, 3, 4). The tyrosine kinase inhibitor imatinib mesylate is approved for first- line treatment of advanced GIST. as well as for adjuvant treatment following resection of KIT-positive GIST (5). Despite the early clinical success of imatinib for the treatment of GIST, it is rarely curative, with the main outcome being partial response or stable disease, requiring lifelong therapy (6). In addition, imatinib re- sistance frequently arises from second-site mutations in Kit (7, 8).We previously generated a mouse model of GIST that carries the KitV558Δ mutation, which is prototypical for Kit exon 11 mu- tations found in both spontaneous and familial GIST (9, 10). We also engineered imatinib-resistant KitV558Δ;T669I/+ mice thatmodel the human “gatekeeper mutation” KitT670I (7, 8, 11). Both mouse models develop gastric and colonic ICC hyperplasia andcecal GIST by histology and aberrant activation of SRC family kinase (SFK), PI3-kinase (PI3K)/AKT/S6, STAT3, and MAPK/ ERK signal transduction pathways indistinguishable from human GIST (10–13). While these findings confirm the central and per- sistent role of KIT in the pathogenesis of GIST, they also highlightthe need to identify other targets to improve clinical outcomes per se and provide the means to prevent or overcome the emergence of resistant clones.

As aberrant KIT signaling is essential for most GISTs, we aimed to identify the proximal downstream targets critical for tumor development and/or maintenance.Kit gain-of-function mutations are also found in neoplasms of the hematopoietic system, such as in mast cells and myeloid cells. In contrast to Kit mutations in GIST, which typically are weak gain- of-function mutations, Kit gain-of-function mutations in the he- matopoietic system are strong mutations typically arising in theactivation loop of the Kit kinase. This appears to be the result of the activation by KIT of a negative regulator of PI3K, SHIP1, in hematopoietic lineages. In agreement with this idea, SHIP1 loss-of- function mice display mast cell hyperplasia (14).The receptor tyrosine kinase KIT contains 22 cytoplasmic tyro-sine phosphorylation sites that are conserved between mouse and humans. Normal and oncogenic kinase activity of KIT results intheir phosphorylation and the recruitment of a multitude of signal transducers to one or more of these docking sites on KIT. Depending on the cellular context, this signal transduction process elicits various biological responses, such as cell survival, proliferation, secretion, and migration. Two phosphorylation sites have been extensively studied in the nononcogenic setting. The phospho-Y567 (pY567) site, among other signaling intermediates, interacts with the SFKs (15, 16),STAT3, and GRB2 (17), while phospho-Y719 (pY719) is the sole site on KIT for the recruitment of PI3K family members (18, 19).We and others have studied the individual contributions of pY567 and pY719 to nononcogenic KIT signal transduction and, ultimately, cellular fate decisions using knock-in mouse models.

Generally, KIT is essential for melanogenesis, hematopoiesis, and gametogenesis. KitY567F/Y567F mice specifically lacking the KITY567 phosphorylation site exhibit minor pigmentation changes and an age-dependent differentiation block in pro-B and pro-T cells, anerythroblast-intrinsic defect in stress erythropoiesis, but normal fertility (20–22). In contrast, KitY719F/Y719F mice lacking the KITY719 phosphorylation site have normal pigmentation and hematopoiesis, but males are sterile and females have defective ovarian folliclematuration (20, 23–25). Importantly, while KIT signaling is re- quired in the ICC lineage, KIT-mediated SFK or PI3K signaling isdispensable, since both KitY567F/Y567F and KitY719F/Y719F mice show normal ICC development (4, 22, 26).The individual downstream KIT signaling pathways required for the oncogenic transformation of ICCs and GIST maintenance are unknown, however. Identification of vital pathways could provide additional strategies to enhance the efficacy of existing therapies orto circumvent imatinib resistance. Thus, double–knock-in mouse models were generated combining the oncogenic KitV558Δ mutation with either the KitY567F or the KitY719F mutation in the endogenousKit locus. Vital pathways identified with this approach were then targeted by specific inhibitors in both our imatinib-sensitive and imatinib-resistant mouse models to translate the genetic findings.

Results
PI3K, but Not SFK, Is Required for ICC Hyperplasia and GIST Oncogenesis. To dissect the in vivo contribution of individual signal transduction pathways emanating from oncogenic KIT, we genetically abrogated the SFK-binding site pY567 or the PI3-kinase binding site pY719 inthe KITV558Δ oncoprotein by substituting the tyrosine to phenyl- alanine (Fig. 1A and Fig. S1). Heterozygosity for the KitV558Δ allele is sufficient to induce GIST development, while homozygositycauses perinatal lethality in mice (10). Because KIT dimerizes, homozygous phenylalanine substitutions were necessary to abro- gate signaling from the specified tyrosines. KitV558Δ;Y567F/Y567F mice were viable and had longer survival than KitV558Δ/+ and KitV558Δ;T669I/+ mice, while KitV558Δ;Y719F/Y719F mice had a normal lifespan (Fig. 1B). KitV558Δ;Y567F/Y567F mice developed GIST in the cecum that was histologically similar to KitV558Δ/+ tumors, but significantly smaller. KitV558Δ;Y567F/Y567F mice had less ICC hyperplasia in the stomach and colon (Fig. 1C, compare d and f with g and i) and less distention of the ileum from tumor obstruction, likely accounting for their longer survival (Fig. 2 A–C). Meanwhile, KitV558Δ;Y719F/Y719Fmice (n = 14) up to 29 mo of age did not develop GIST. Histologicalanalysis revealed normal morphology of the stomach, cecum, and colon, with a normal density of ICCs in the myenteric plexus (Fig. 1C, j–l).KitV558Δ;Y567F/Y567F Tumors Have Disrupted SFK Signaling. While KitV558Δ;Y567F/Y567F mice had a smaller tumor and lived longer than KitV558Δ mice, KitV558Δ;Y567F/+ mice with a wild-type (WT) Kit allele had a shorter lifespan than KitV558Δ;Y567F/Y567F mice, indicating that one dose of KITY567 signaling affected survival (Fig. 2D). Simi- larly, increasing the oncogenic dose in KitV558Δ;Y567F/V558Δ ;Y567F mice further reduced survival despite disruption of KITY567 sig- naling, confirming that signaling from other KIT tyrosines con- tributed to KIT signaling in GIST (Fig. 2D).

To verify the impaired SFK signaling in KitV558Δ;Y567F/Y567F tu- mors, we performed IHC and Western blot analysis. KIT staining was uniformly high in KitV558Δ/+ and KitV558Δ;Y567F/Y567F tumors (Fig. 2E, b and c), while adjacent mucosal tissues, lymphoid fol- licles, and remaining intervening fibers of smooth muscles lacked KIT staining, as expected. Notably, KitV558Δ;Y567F/Y567F tumors were more heterogeneous, with remaining muscle fibers and fi- brotic areas present. Importantly, S6 phosphorylation, a down- stream indicator of PI3K activation, was equally high in KitV558Δ/+ and KitV558Δ;Y567F/Y567F tumors (Fig. 2E, e and f). In contrast, phos- phorylation of MAPK, and toa lesser extent STAT3, was diminished in KitV558Δ;Y567F/Y567F GIST lesions (Fig. 2E, h, i, k, and l). Imatinib treatment of KitV558Δ/+ mice for 6 h reduced S6, MAPK1/3, and(A)Gross pathology of the gastrointestinal tract of a 3-mo-old KitV558Δ;Y719F/Y719F mouse that appeared normal without cecal GIST, megaileum, or megacolon in comparison with KitV558Δ/+ and KitV558Δ;Y567F/Y567F mice (10, 11) (Fig. 2A). (B) Immunohistochemical KIT staining of ICCs in cross-sections of the flat-mounted cecum from 7-mo-old WT and KitV558Δ;Y719F/Y719F mice (n = 14; 3–29 mo old). The typical presence and distribution of individual ICCs in the plane of the myenteric plexus (ICC-MY, arrow) between the circular (CM) and longitudinal (LM) muscle layers, as well as intramuscular ICC (ICC-IM; arrowhead), arepreserved in KitV558Δ;Y719F/Y719F mice. Note the increased tissue surface in the right section due to the tangential cut of the sample, but the absence of ICC hyperplasia. n = 3. (Scale bar: 100 μm.) (C) Kaplan–Meier survival plot showing similar survival of KitV558Δ;Y719F/Y719F, KitV558Δ;Y719F/+, and KitV558Δ;Y719F/V558Δ;Y719F mice. The median survival of KitV558Δ;Y719F/Y719F was 28 mo, as shown in Fig. 1. (D)

Representative H&E-stained sections of testis showing normal morphology and spermatogenesis in 6-mo-old WT and KitV558Δ;Y719F/+ mice and empty tubules in testis of 6-mo-old KitV558Δ;Y719F/Y719F and 3-mo-old KitV558Δ;Y719F/V558Δ;Y719F mice. n = 3 each. (Scale bar: 50 μm.) (E) Kinase activity of WT KIT, KITV558Δ, and KITV558Δ;Y719F mutants using the PathHunter eXpress receptor tyrosine kinasefunctional assay. Quantitative detection of β-gal activity (bioluminescence) is reported as arbitrary units. (F) Western blots from 293T cells transfected withcontrol empty vector (Ctrl vector), WT Kit, KitV558Δ, or KitV558Δ;Y719F/Y719F or cotransfected with KitV558Δ and WT or KitV558Δ;Y719F/Y719F and WT. (G) Immunoblot analysis of bone marrow-derived mast cell extracts from WT, KitV558Δ/+, and KitV558Δ;Y719F/+ mice. Antibodies specific for pKIT-Y567/Y569, pKIT-Y719, and KIT were used.STAT3 phosphorylation without altering KIT expression (Fig. 2E, a, d, g, and j), consistent with our previous studies (11–13). In contrast, MAPK1/3 phosphorylation was at an intermediate level in untreated KitV558Δ;Y567F/Y567F tumors, indicating that SFK partially contributed to MAPK activation (Fig. 2E, g–i).Western blot analysis further confirmed similar PI3K pathwayactivation in untreated KitV558Δ;Y567F/Y567F and KitV558Δ/+ tumors, as demonstrated by phosphorylation of S6 and 4E-BP1 (Fig. 2 F and G). Again, phospho-MAPK1/3 (ERK1/2) was reduced in KitV558Δ;Y567F/Y567F tumors. SFK pathway activation was greatly reduced, as expected, but phospho-STAT3 was greatly reduced as well (Fig. 2 F and G). Taken together, our findings indicate that the oncogenic KitV558Δ mutation combined with the KitY567F mutation did not affect PI3K signaling, but reduced the MAPK and STAT3 pathways and almost abrogated SFK activation.Kit Kinase Is Functional in KitV558Δ;Y719F/Y719F Mice. The gastrointes- tinal tract in KitV558Δ;Y719F/Y719F mice appeared normal, with normal stomach, cecum, and colon morphology.

KIT immuno- histochemistry revealed normal morphology and density of myenteric and intramuscular ICCs (Figs. 1C, j–l and 3 A and B).The absence of GIST in KitV558Δ;Y719F/Y719F mice explained theirnormal lifespan (Fig. 1B). Remarkably, KitV558Δ;Y719F/+ mice (harboring one WT Kit allele) also had a normal lifespan and did not develop GIST (Fig. 3C), which was surprising, given our assumption that mutant KIT protein could form heterodimers with WT KIT (KIT+-KITV558Δ;Y719F) and activate the PI3Kmoiety of WT KIT. Furthermore, KitV558Δ;Y719F/V558Δ;Y719F micehomozygous for both mutations had an extended lifespan and failed to develop GIST. These results suggest that PI3K signaling is critical for GIST oncogenesis, and that even partial abrogation of PI3K recruitment to KIT is sufficient to prevent tumorigen- esis, regardless of KIT oncogene dosage. Thus, PI3K may be a desirable candidate for pharmacologic targeting without the re-KIT activity and similar to that of the KITV558Δ mutant (Fig. 3E). Both mutants were hypersensitive to increasing doses of KitL compared with WT KIT.To further confirm that KITV558Δ;Y719F is functional, and to assess whether it is capable of canonical transphosphorylation, we transfected Kit constructs containing the WT, V558Δ, or V558Δ; Y719F mutations into 293T cells and measured KIT autophos- phorylation by detection of specific KIT phosphotyrosine residues. The WT KIT receptor showed weak Y719 phosphorylation andbarely detectable Y567/9 phosphorylation, similar to empty vector (Fig. 3F), consistent with the absence of KitL. The weak phos- phorylation of Y719 was most likely due to KIT overexpression leading to random dimerization of receptors.

In contrast, the KITV558Δ mutant had a net increase in both Y719 and Y567/9phosphorylation, in accordance with its constitutive kinase activity.As expected, the KITV558Δ;Y719F mutant lacked Y719 phosphory- lation, but exhibited levels of Y567 and Y569 phosphorylation comparable to those of the KITV558Δ mutant (Fig. 3F), indicating functional kinase activity of KITV558Δ;Y719F. Cotransfection of KITV558Δ;Y719F with WT KIT showed weak phosphorylation of Y719 in addition to Y567/9, confirming that KITV558Δ;Y719F has canonical kinase activity in trans. In this combination, only WTKIT possessed a Y719 site, and its phosphorylation, at least the lower band, resulted from the presence of the mutantKITV558Δ;Y719F, since Y719 phosphorylation of the lower band did not occur in the “WT-only” KIT lane.To confirm that KITV558Δ;Y719F is functional when expressedat endogenous levels, primary cultures of bone marrow-derived mast cells (BMMCs) were derived from WT, KitV558/+, and KitV558Δ;Y719F/+ mice (Fig. 3G). At baseline, the WT and mu-tant KIT proteins were barely phosphorylated at KITY719 and KITY567/569. The addition of KitL increased phosphorylation to agreater degree in the mutants than in the WT, indicating that the mutants are hypersensitive to KitL stimulation and essentiallyquirement for complete Kit inhibition. Therefore, the absence of GIST in KitV558Δ;Y719F/Y719Fmight rescue azoospermia. We found that KitV558Δ;Y719F/Y719F mice recapitulated the testis phenotype of KitY719F/Y719F mice. Matings with multiple WT females over more than 6 mo did not produce offspring, testicular volume was reduced, and testis cross-sections revealed empty tubules (Fig. 3D).

As was the case with survival, increasing the oncogenic dose in KitV558Δ;Y719F/V558Δ;Y719F mice was not sufficient to compensate for the loss of KITY719, indicating the need for at least one Y719 phosphorylation site for spermato- genesis. KIT is also known to have roles in hematopoiesis and mast cell development, and Kit mutant mice often present with a blood and/or mast cell phenotype. Skin mast cell numbers are increased in KitV558Δ mice, while KitV558Δ;T669I/+ mice develop mast cell hyperplasia and microcytic erythrocytosis (10, 11). Neverthe- less, in this study, the KitV558Δ;Y719F/Y719F mice had normal pe- ripheral blood and skin mast cell levels.Even though KitV558Δ is a potent oncogene and KitY719F only affects PI3K activation in mice, we cannot exclude the possibility that when both KitV558Δ and KitY719F mutations reside on the same allele, the protein does not fold properly, thereby inacti- vating KIT (10, 24). However, since KitV558Δ;Y719F/Y719F mice have normal ICCs and pigmentation, it is likely that KIT is functional. Given that Kit-null is perinatal lethal (27), the strongest in vivo evidence that this combination does not cause any severe KIT functional defects is our ability to derive viable adult mice ho- mozygous for the KitV558Δ;Y719F allele. To confirm that the KIT kinase is functional in the KitV558Δ;Y719F mutation, we performed an in vitro kinase assay using cells expressing full-length KITmutants. At baseline, without the addition of KIT ligand (KitL), KITV558Δ;Y719F kinase activity was significantly higher than WTmice is not due to a defect of the double-mutant KIT kinase, butrather must be explained by the lack of KIT pY719 signaling.Response to Pharmacologic Inhibition of PI3K and MAPK in KitV558Δ/+ Mice. Because PI3K signaling from KIT was required for GIST formation, we investigated whether selective pharmacologic in-hibition of PI3K was effective against established tumors in KitV558Δ/+ mice. We first tested the broadly acting dual PI3K/ mTOR inhibitor voxtalisib (XL765). Tumor lysates from mice treated for 6 h showed decreased PI3K signaling, manifested by reductions in pAKT, pS6, and p4EBP1 (Fig. 4A). KIT and STAT3 phosphorylation were not affected, but, surprisingly, MAPK phosphorylation was increased. Seven days of treatment reduced tumor cell proliferation by 70% (Fig. 4B), although the histological response was minimal (Fig. 4C, c). To counteract MAPK activation by voxtalisib, we sought to use a MAPK in- hibitor. The MEK inhibitor PD-325901 inhibited MAPK sig- naling without affecting PI3K and STAT signaling, but did not alter tumor proliferation (Fig. 4 B and D). Treatment with vox- talisib and PD325901 for 6 h reduced both PI3K and MAPK signaling, as well as phospho-STAT3 signaling (Fig. 4E). How- ever, toxicity during 7 d of therapy required dosage reductions of both drugs to 5 mg/kg.

Notably, low-dose voxtalisib and PD325901 therapy reduced tumor cell proliferation to a comparable degree as high-dose voxtalisib monotherapy (Fig. 4B), although the his- tological response was minimal (Fig. 4C, d). Taken together, these results indicate that targeting PI3K with the dual PI3K/mTOR in- hibitor voxtalisib reduces tumor cell proliferation, but up-regulation of MAPK signaling might limit its efficacy.The MAPK activation with voxtalisib therapy likely results from a feedback loop due to mTOR inhibition (28). Therefore, to avoid MAPK activation, we next assessed the pan-class I PI3K selectiveinhibitor pilaralisib (XL147), which does not inhibit mTOR and thus more closely resembles the genetic abrogation of PI3K sig- naling from KIT. Tumor lysates from KitV558Δ/+ mice treated for4 h with pilaralisib showed decreased PI3K signaling with reduced pAKT, pS6, and p4EBP1 (Fig. 5A). In contrast, phosphorylated KIT, STAT3, and MAPK were unaffected (Fig. 5 A and B). IHC of tumors following 7 d of pilaralisib treatment revealed a lack of phosphorylated S6, no effect on MAPK1/3 or STAT3 activation (Fig. 5C), and reduced proliferation, yet only a minimal histological response (Fig. 4 B and C, e).Because KitV558Δ;Y567F/Y567F tumors have less MAPK signaling and are smaller than KitV558Δ/+ tumors, we reasoned that pilar- alisib might be more effective if combined with a MAPK inhib- itor. Thus, we treated KitV558Δ/+ mice with the MEK inhibitor PD-325901 in addition to pilaralisib. After 6 h of treatment,PI3K and MAPK signaling were reduced, as was STAT3 sig- naling (Fig. 5D). Unfortunately, combination therapy was too toxic over 7 d of therapy.Since voxtalisib and pilaralisib in combination with PD-325901 were not well tolerated, we evaluated other PI3K and MEK in- hibitors. Alpelisib is a potent PIK3CA (PI3K-α) inhibitor, withminimal effects on PIK3CB (PI3K-β), PIK3CD (PI3K-δ), orPIK3CG (PI3K-γ). KitV558Δ/+ mice treated for 7 d with alpelisib had decreased PI3K signaling with reduced pAKT, pS6, andp4EBP1, while KIT, MAPK, and STAT3 activation were unchanged (Fig. 6A). Mice treated for 7 d with the ATP noncompetitive MEK inhibitor binimetinib had less pMAPK1/3 (Fig. 6B).

pSTAT3 was also reduced, but PI3K signaling was unaltered. The com- bination of alpelisib and binimetinib was well tolerated and re- duced PI3K pathway activation, pMAPK1/3, and pSTAT3 (Fig. 6C). Furthermore, tumor cell proliferation was reduced by 75%, while the histological response was slightly improved (Fig. 4 B and C, f).Imatinib-Resistant KitV558Δ;T669I/+ Tumors Respond to Dual PI3K and MEK Inhibition. Since alpelisib and binimetinib were found to target two major signaling pathways downstream of KIT and to lack toxicity in KitV558Δ/+ mice, we evaluated whether they couldbe used in combination to overcome imatinib resistance. For this purpose, imatinib-resistant KitV558Δ;T669I/+ mice (11) were treated for 7 d with alpelisib and binimetinib. Both PI3K and MAPK signaling were reduced, as was tumor cell proliferation (Fig. 7 A and B). Furthermore, the histological response was increased (Fig. 7C). Thus, targeting downstream components of KIT sig- naling was also effective in the setting of imatinib resistance.DiscussionHere we engineered double-mutant mice carrying both the on- cogenic KitV558Δ mutation and the KitY567F or the KitY719F mu- tation on the same allele. Mice carrying the KitV558Δ;567F allele developed smaller GIST lesions than the KitV558Δ mutant mice, indicating that signal transducers that bind to KITY567 have a role in tumor growth. By crossing KitV558Δ;567F/+ mice with our previously developed single-mutant KitY567F/+ mice, we gener- ated double-mutant compound heterozygous KitV558Δ;Y567F/Y567F mice that are homozygous for the Y567F mutation, to ensure that any signal that normally emanates from phospho-Y567 is abrogated. SFK activation was dramatically impaired in GIST of the KitV558Δ;Y567F/Y567F mice compared with the KitV558Δ/+ mice. This is consistent with an important role for SFKs in KIT- mediated activation of the RAS/MAPK pathway (15).

In addi- tion, we observed a partial reduction in STAT3 signaling in tumors of KitV558Δ;Y567F/Y567F mice (29). In mast cell/myeloid cell transformation by oncogenic Kit, Y567F mutation accelerated tumorigenesis, presumably due to inhibited activation of SHIP1,a hematopoietic-specific negative regulator of PI3K (14, 17, 30). In contrast, in KitV558Δ;Y567F/Y567F mice, the Y567F mutation dimin- ished tumorigenesis, indicating that KITpY567 is a positive regulator of tumorigenesis in GIST. Since a mild hematopoietic pro-B/pro T-cell phenotype in KITY567F/Y567F mutant mice becomes apparent after age 8 mo, biochemical analysis of tumor tissue was performed in tumors from 3- to 4-mo-old mice (20, 22).Importantly, our results provide in vivo evidence that the di- rect activation of the PI3K-pathway via KITY719 is required for GIST tumorigenesis. The KITY719 phosphorylation site also has been postulated to be of importance in myeloproliferative dis- ease induced by the kinase domain mutation KITD814V (human KITD816V), based on experiments with chimeric proteins con- sisting of the extracellular domain of human M-CSFR and the intracellular domain of murine KIT (18, 31). We considered the possibility of defective ICC development in KitV558Δ;Y719F/Y719F mice, which would readily explain the absence of hyperplasia and tumor development, as ICCs or their progenitors are the pre- sumed cells of origin of GIST (32). However, ICCs developednormally and stained positive for KIT. In addition, the kinase activity of the KITV558Δ;Y719F protein was not impaired, strength- ening the idea that disruption of PI3K signaling is responsible for the lack of tumor development. The mutant KIT alleles were expressed under the control of the endogenous Kit transcriptionmachinery and thus expressed continuously during ICC devel- opment; that is, the experiments did not distinguish whether the phenotypes derived were due to a failure in tumor initiation or a failure in maintaining an oncogenic state.

Also, KitV558Δ;Y719F/Y719F mice lacked the PI3K-binding site in all KIT-expressing cells starting at fertilization. Thus, we cannot rule out that the absence of tumorigenesis in our model could be the result of a defect in tumor initiation.In addition, it is possible that proteins other than PI3K-family members are being recruited to the phospho-KITY719 site and are necessary for transformation. Nevertheless, pharmacologic inhibi- tors of the PI3K pathway in tumor-bearing KitV558Δ/+ mice, in- cluding the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3Kinhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpeli- sib, each diminished tumor proliferation, indicating that PI3 kinase makes a major contribution to tumor cell proliferation in estab-lished GIST. The presumed selectivity of these inhibitors for PI3K was generally confirmed in our mice, since phosphorylation of S6 kinase and 4EBP1 were inhibited, while MAPK and STAT3 activation were unaffected. The dual PI3K/mTOR inhibitor voxta- lisib increased MAPK activation in GIST. This is consistent with reports that mTORC1 inhibition promotes MAPK activation in a mouse model of prostate cancer and in patients with metastatic disease subjected to mTOR inhibition therapy (28). The feedbackactivation of MAPK with voxtalisib provided a rationale for using an MEK inhibitor in combination with a PI3K inhibitor.In contrast to mice carrying the KitV558Δ;Y719F/Y719F double mu- tation, mice carrying a KitV558Δ;Y567F/Y567F double mutation de- veloped GIST, although tumor development was attenuated and the animals had an extended lifespan.

In these animals, PI3K sig- naling was not affected, but SFK pathway activation was strongly diminished, and phospho-MAPK1/3 and phospho-STAT3 were reduced as well. In agreement with those observations, treatment of KitV558Δ/+ mice with the MEK inhibitors PD-325901 or binimetinib diminished MAPK1/3 and STAT3 phosphorylation/activation. These findings indicate a requirement for the direct engagement of distinct pathways by KIT for full tumor growth. Thus, dual in- hibition of both the PI3K and MAPK branches in oncogenic KIT signaling was pursued. Whereas combination treatment with either voxtalisib plus PD-325901 or pilaralisib plus PD-325901 was too toxic, combination treatment with the PI3K-alpha inhibitor alpelisib and the MAPK inhibitor binimetinib was tolerated and shows pro- mise for use in patients with GIST, particularly those with imatinib- resistant GIST, as demonstrated in imatinib-resistant gatekeeper KitV558Δ;T669I/+ mice. PI3K inhibitors alone and in combination with imatinib have shown antitumor effects in GIST xenograft models;however, the most common imatinib-resistant GIST mutations (KIT exons 11–13 and 11–14) have not been tested (33, 34). Tar- geting of the PI3K pathway with the AKT inhibitor MK-2206 has been shown to increase imatinib efficacy in cell culture and in xe- nografts with imatinib-sensitive and -resistant GIST cell lines (35).Our studies with immunocompetent mice carrying either the KitV558Δ or the common KIT imatinib-resistant gatekeeper mutation strengthen our argument that the combination of PI3K and MEK inhibition could be beneficial for treating patients with imatinib- resistant GIST.

In summary, our results suggest that a detailed analysis of dis- tinct oncogenic signaling pathways mediated by oncogenic tyrosine kinases in specific cellular contexts may lead to new and improved treatment strategies.Generation of Mouse Strains. The V558Δ mutation was introduced by site- directed mutagenesis into a 2.1-kb EcoRI/MluI fragment across Kit exons 9– 11 from a 129/SvJ mouse library (24) serving as the 5′ homology arm of the targeting vector. For the KitV558Δ;Y567F allele, the Y567F mutation was in- troduced into the same 5′ arm for homologous recombination (HR) (Fig. S1). The 5′ HR arm was linked 3′ to a floxed neomycin-resistance (NEO) gene expression cassette. The 3′ HR arm for the KitV558Δ;Y567F allele was a 1.3-kb MluI/BsrGI-NcoI fragment across exons 12–13 (Fig. S1). For the KitV558Δ;Y719F allele, this arm was elongated by the 3′ directly neighboring 129/SvJ geno- mic DNA, a 2.1-kb BsrGI-NcoI/BamHI fragment including exon 14, and a 3.8-kb BamHI fragment across exons 15–17 including the Y719F mutation in exon 15. The final targeting vector was completely sequenced before linearization and electroporation into CJ7 ES cells (36).Screening of BamHI-digested genomic DNA from 288 ES cell clones each by Southern blot analysis with a 3′ external probe across Kit exon 14 or a 5′ external probe across Kit exon 8 yielded 17 (5.9%) and 19 (6.6%) positive clones for the KitV558Δ;Y567F and KitV558Δ;Y719F projects, respectively. Four ES cell clones carried both the V558Δ mutation and the nearby Y567F muta- tion, and two clones carried both the V558Δ mutation and the distant Y719F mutation, respectively, as assessed by sequencing, and showed a normal karyotype. C57BL/6J blastocyst injections of two of these clones each gave rise to 19/21 high-grade chimeras (>90% agouti coat color), one each of which gave germ line transmission of the double mutations. After crossing to C57BL/6J mice, in all agouti F1 animals heterozygous for the NEO allele, the presence of the V558Δ;Y56F and V558Δ;Y719F mutations, and the integrity of both loxP sites was confirmed by sequencing. To remove the floxed NEO cas- sette, F1 KitV558Δ;Y567F-NEO/+ and KitV558Δ;Y719F-NEO/+ males were bred to B6.FVB- Tg(EIIa-cre) C5379Lmgd/J females MEK162 (The Jackson Laboratory).