Results

Derivation and Phenotypic Characterization of KitV558Δ;T669I/+ Gatekeeper Mice.

To investigate the consequences of second-site KIT mutations on imatinib susceptibility and GIST development in vivo, we generated a mouse model introducing both the Kit^V558Δ and the Kit^T669I gatekeeper mutation, corresponding to human KIT^T670I and

found in cases of imatinib-resistant GIST, into the endogenous Kit locus. To facilitate simultaneous introduction of the two point mutations into the mouse Kit gene, the tar-geting vector included a floxed neomycin-resistance gene (NEO) cassette in Kit intron 11 for positive selection of recombinant ES cells containing both the V558Δ (exon 11) and T669I (exon 14) mutations (Fig. 1A). After successful integration and germ-line transmission of the KitV558Δ;T669I-NEO allele, the intronic NEO cassette was removed by crossing to Tg(EIIa-cre) mice (19). The resulting KitV558Δ;T669I allele retains a single loxP site in intron 11 (Fig. 1A3). A Kit^V558Δ allele with a loxP site in intron 11 was gen-erated as a control for the KitV558Δ;T669I allele (Fig. S7A).

Fig. 1: Derivation and phenotypic characterization of Kit-gatekeeper mice.

(A) Targeting strategy for the simultaneous knock-in of V558Δ (exon 11) and T669I (exon 14) into the 129/Sv Kit locus. Blue and red bars denote the exons with the respective point mutations. A similar tar-geting vector with a shorter 3′ homology arm was used to generate the single-mutant Kit^V558Δ/+ mice (Fig.

S7A). Triangles (not drawn to scale) indicate loxP sites; white gaps indicate BamHI restriction sites; bar indicates 518-bp Southern blot probe. DTA, diphtheria toxin A gene; NEO, neomycin resistance gene. (B) Kaplan-Meier survival plot showing increased survival of gatekeeper-mutant KitV558Δ;T669I/+ mice in com-parison with Kit^V558Δ/+ mice (n ≥ 43 each; ticks indicate censored subjects). (C) Photographs of ileocecal junctions showing reduced length and diameter of tumor alongside the cecum in KitV558Δ;T669I/+ mice (Middle; red bracket indicates straight cecal GIST) in comparison with Kit^V558Δ/+ mice (Bottom; blue bracket indicates twisted cecal GIST). Of note, the cecum is significantly shorter in KitV558Δ;T669I/+ mice than in wild-type mice (Top; black arrow) and Kit^V558Δ/+ mice. Representative pictures of 3-mo-old ani-mals are shown with colon facing down left and ileum facing down right (n ≥ 59 each). (Scale bar, 1 cm.)

Double-mutant KitV558Δ;T669I/+ mice are viable and fertile but, in contrast to Kit^V558Δ/+

mice, were born at sub-Mendelian ratios when crossed to wild-type mice (35% instead of 50% heterozygous offspring). In comparison with single-mutant Kit^V558Δ/+ mice, dou-ble-mutant KitV558Δ;T669I/+

mice had a prolonged lifespan with a median survival of 14 mo (n > 43 each, P < 0.0001) (Fig. 1B).

Invariably, KitV558Δ;T669I/+

mice developed cecal tumors. These tumors were smaller than in Kit^V558Δ/+ mice, perhaps explaining the improved survival by a decreased chance

of intestinal obstruction (Fig. 1C). The average tumor diameter in 3-mo-old animals was fivefold smaller in KitV558Δ;T669I/+ than in Kit^V558Δ/+ mice (1.4 ± 0.1 mm vs. 7.0 ± 0.3 mm, P < 0.001). Interestingly, not only were the cecal tumors smaller, but the length of the cecum was significantly shorter in KitV558Δ;T669I/+ mice compared with Kit^V558Δ/+ and wild-type mice (13 ± 2 mm vs. 24 ± 2 mm, P = 0.003) (Fig. 1C).

Fig. 2: Cecal GIST and pronounced gastric and colonic ICC hyperplasia in KitV558Δ;T669I/+ mice.

Cross-sections of stomach (A–C; higher magnification is shown in A*–C*), cecum (D–F), and colon (G–

I) of 3- to 4-mo-old wild-type, Kit^V558Δ/+ and KitV558Δ;T669I/+ mice. Arrows indicate normal thin layer of myenteric ICC in wild-type samples. ICC hyperplasia in stomach and colon samples is indicated by black bars. Note the extensive hyperplasia involving the circular muscle layer (dotted lines) in the stomach of KitV558Δ;T669I/+ mice. Photographs show representative H&E staining; n ≥ 3 each. (Scale bars: 50 μm in A*–C*; 100 μm in A–I.)

Histological analysis of the shortened cecum of the KitV558Δ;T669I/+

mice revealed an in-tact lumen and mucosa similar to those in Kit^V558Δ/+ mice. In contrast to the overall re-duction in tumor size in the cecum, KitV558Δ;T669I/+ mice developed more pronounced ICC hyperplasia in the stomach (Fig. 2C and I; also see Fig. S9A a–d) and colon than Kit^V558Δ/+ mice (Fig. 2B and H; also see Fig. S9B a–d).

Immunohistochemical analysis of gastric and colonic sections revealed KIT staining as well as phosphorylation of S6, MAPK, and STAT3 in the ICC hyperplasia of Kit^V558Δ/+ as well as KitV558Δ;T669I/+ mice (see Fig. S9A e–n and B e–n), but no significant differences in signal transduction were apparent in the two strains that could explain the exacerbated ICC hyperplasia in KitV558Δ;T669I/+ mice. H&E-stained sections of cecal tu-mor lesions in both Kit^V558Δ/+ and KitV558Δ;T669I/+ mice showed a histology indistinguish-able from human GIST (Fig. 2E and F).

The KitV558Δ;T669I/+ Gatekeeper Mutation Confers Resistance to Imatinib and Dasatinib in Vivo.

To investigate the in vivo sensitivity of the KitV558Δ;T669I gatekeeper mutation to tyrosine kinase inhibitors (TKIs) with KIT inhibitory potential, we treated cohorts of 3- to 4-mo-old KitV558Δ;T669I/+

mice with imatinib, dasatinib, sunitinib, or sorafenib. First, we ana-lyzed signaling cascades known to be affected by imatinib after short-term (6-h) drug treatments (Rossi et al. 2006; Rossi et al. 2010). Second, proliferation, apoptosis, and histological changes within tumors and adjacent mucosa were assessed after long-term (7-d) treatment with KIT inhibitors. In addition, changes in the phosphorylation status of proteins in GIST following long- term treatment were evaluated. Age-matched sin-gle-mutant Kit^V558Δ/+ mice were treated analogously to serve as controls.

To examine if the KIT kinase in KitV558Δ;T669I/+ mice is sensitive to inhibition by imatinib, tumor lysates of individual mice were subjected to Western blotting with phospho-Y719-KIT and KIT antibodies. In tumors of imatinib-treated Kit^V558Δ/+ control mice, KIT phosphorylation was inhibited, as reported earlier (Fig. 3A) (Rossi et al.

2006; Rossi et al. 2010). In contrast, in tumors of KitV558Δ;T669I/+ mice, KIT phosphoryla-tion was unchanged after treatment with imatinib (Fig. 3B). KIT inhibiphosphoryla-tion could be re-stored by treatment with sunitinib, which diminished KIT phosphorylation to similarly low levels in both Kit^V558Δ/+ and KitV558Δ;T669I/+ mice (Fig. 3A and B).

Fig. 3: The KitV558Δ;T669I double mutation confers resistance to imatinib in vivo. Sunitinib over-comes resistance.

(A and B) Tumor protein lysates from individual Kit^V558Δ/+ (A) and KitV558Δ;T669I/+ (B) (Left) animals treated for 6 h with vehicle (Ctrl), imatinib (IM), or sunitinib (SU) were subjected to Western blotting with phospho-Y719-KIT and KIT antibodies. Representative blots are shown. (Right) The ratio of phosphory-lated to total KIT in GISTs of KitV558Δ;T669I/+ mice after imatinib and sunitinib treatment was quantified by densitometry (n = 3–5 animals per treatment group; error bars indicate means ± SD). (C) Quantification of phospho-STAT3⁺ nuclei per field (cf. D s–x and corresponding explanations). n = 3 each; error bars indicate means ± SD. (D) Representative results of IHC analysis with antibodies specific for KIT (a–f), phospho-S6 ribosomal protein (g–l), phospho-MAPK (m–r), and phospho-STAT3 (s–x) on sections of GISTs from single- or double-mutant mice treated for 6 h with vehicle, imatinib, or sunitinib. Tumor sec-tions from different treatment groups and genotypes (n ≥ 3 each) were placed next to each other on the same microscopy slide to enable cross-comparison of staining intensities. (Scale bars: 50 μm.)

Because of the small size of neoplastic lesions in KitV558Δ;T669I/+

mice, the effect of drug treatment on downstream signaling networks was examined primarily by using an immunohistochemical (IHC) approach. As is characteristic for most human GIST, tu-mors stained positive for KIT independent of the drug used or treatment duration (Fig.

3D a–f and Fig. 4D a–d and Fig. S8A a–f and B a–f). Tumors of control (vehicle-treated) animals of both genotypes showed strong phosphorylation of ribosomal protein S6, MAPK, and STAT3 (Fig. 3C and D g, h, m, n, s, and t). After 6 h of treatment with imatinib, phosphorylation of these signaling components was strongly reduced in tu-mors of Kit^V558Δ/+ animals (Fig. 3C and D i, o, and u) but not in tumors of animals with the KitV558Δ;T669I mutation (Fig. 3C and D j, p, and v).

The latter result is of particular note, because imatinib has the potential to inhibit multiple kinases (e.g., ABL and PDGFR) that might be expressed and activate these targets in GIST parallel to or downstream of KIT. Furthermore, in vitro assays with the

intracellular kinase domain of KIT had demonstrated that imatinib inhibits wild-type KIT as efficiently as KIT^V558Δ (Karaman et al. 2008). It is not known whether KIT heterozygosity (i.e., the coexpression of one mutant and one wild-type KIT allele), as detected in the GISTs of most patients, results in the formation of functional KIT heter-odimers driving oncogenic signaling. Assuming that imatinib inhibits wild-type KIT and possibly other kinases expressed in our heterozygous KitV558Δ;T669I/+ mice, we de-duce that wild-type KIT and off-target kinase inhibition is not sufficient to affect GIST signal transduction.

This apparent KITV558Δ;T669I-isoform dependence of phosphorylation of S6, MAPK, and STAT3 also was confirmed when Kit-mutant mice were treated with dasatinib.

Treatment with dasatinib down-regulated phosphorylation of these components of the signal transduction network in GISTs of single-mutant Kit^V558Δ/+ mice (Fig. 4D e, i, and m) but not in GISTs of double-mutant KitV558Δ;T669I/+ mice (Fig. 4D f, j, and n).

Fig. 4: Resistance to dasatinib and sensitivity to sorafenib treatment in KitV558Δ;T669I/+ mice.

(A–C) Tumor lysates from individual Kit^V558Δ/+ and KitV558Δ;T669I/+ animals treated with vehicle, imatinib, sunitinib, dasatinib, or sorafenib were subjected to Western blotting to detect the abundance of phosphor-ylated and total KIT, S6, and MAPK proteins. Representative blots for phospho-Y719-KIT and KIT are shown. The ratio of phosphorylated to total protein was quantified by densitometry. n = 3–4 animals per treatment group; error bars indicate means ± SD. (D) Representative results after short-term treatment with dasatinib and sorafenib; IHC on GIST sections with antibodies as specified in Fig. 3D. (E) Cell pro-liferation in GIST of KitV558Δ;T669I/+ mice is unaffected by long-term treatment with imatinib and dasatinib.

Sunitinib and sorafenib overcome resistance and attenuate cell proliferation. Ki67⁺ nuclei per 250 × 250 μm field; n ≥ 3 each; error bars indicate means ± SD.

These results were confirmed by Western blotting and quantified by densitometry as the ratio of phosphorylated (pY-)S6 to total S6 protein as well as pY-MAPK to total MAPK protein (Fig. 4B and C).

Importantly, the observed resistance at the histochemical and biochemical levels was mirrored in results obtained by long-term treatments: Twice daily treatment for 7 d with imatinib or dasatinib significantly reduced cell proliferation, as determined by Ki67 staining, in GISTs of single-mutant Kit^V558Δ/+ mice (Fig. 4E). In contrast, treatment of double-mutant KitV558Δ;T669I/+ mice with imatinib or dasatinib did not inhibit GIST pro-liferation, nor did it elicit a histological response (Fig. 4E and Tab. 3). Together, these results demonstrate that the sole addition of the gatekeeper mutation in an in vivo model of GIST can cause resistance, as postulated for patients who have imatinib-refractory GIST with a correlating gatekeeper mutation.

Tab. 3: Histological response of GIST lesions in Kit^V558Δ/+ and KitV558Δ;T669I/+ mice to 7-d treatment with imatinib, dasatinib, sunitinib, or sorafenib.

Sunitinib and Sorafenib Overcome Resistance.

Next, we determined the response of the imatinib-resistant KitV558Δ;T669I/+

mice to se-cond-generation TKIs, namely sunitinib and sorafenib, which had been shown in vitro to inhibit cells expressing the KITV558Δ;T669I

mutation (Carter et al. 2005; Guo et al.

2007). Short-term treatment with sunitinib and sorafenib reduced phosphorylation of KIT, S6, MAPK1/3, and STAT3 to similarly low levels in tumors of Kit^V558Δ/+ and KitV558Δ;T669I/+ mice as assessed by IHC (Fig. 3C and D k, l, q, r, w, and x and Fig. 4D g, h, k, l, o, and p) and Western blotting (Fig. 3A and B and Fig. 4A–C). After long-term treatment with sunitinib and sorafenib, KIT-mediated signal transduction and

prolifera-Strain Vehicle Imatinib Dasatinib Sunitinib Sorafenib

Kit^V558Δ/+ None Minimal Minimal Minimal Moderate

None None Minimal None Mild

tion was diminished significantly in GISTs of KitV558Δ;T669I/+

mice to the levels achieved by all four inhibitors (imatinib, sunitinib, dasatinib, and sorafenib) in single-mutant Kit^V558Δ/+ mice (Fig. 4E and Fig. S8A g–x and B g–x). Cell proliferation in tumor- and ICC hyperplasia-adjacent gastrointestinal epithelial cells was not impaired after long-term treatment, indicating no overt toxic side effects of these TKIs in mice at the con-centrations used (Fig. S10C a–i). These experiments demonstrated that the resistance mediated by the KitV558Δ;T669I mutation could be overcome by treatment with sunitinib and sorafenib.

To investigate whether the ICC hyperplasia in the KitV558Δ;T669I/+ mice recapitulated the resistance/susceptibility pattern observed in the cecal neoplastic lesions, gastric cross-sections were examined after 7 d of treatment with imatinib or sunitinib. In con-cordance with our results in cecal tumor lesions, the ICC hyperplasia in KitV558Δ;T669I/+

mice exhibited resistance to imatinib and susceptibility to sunitinib inhibition of KIT signaling and ICC proliferation (Fig. S10C g–x and D), implying that second-generation TKIs also could inhibit the early stages of imatinib-resistant GIST development in KitV558Δ;T669I/+ mice.

Hyperproliferation in Hematopoietic Cell Lineages in KitV558Δ;T669I/+

Mice.

In addition to the pronounced ICC hyperplasia in KitV558Δ;T669I/+

mice in comparison with Kit^V558Δ/+ mice, we observed hyperproliferation in other KIT-dependent lineages.

The mast cell hyperplasia we had observed previously in the dorsal skin of Kit^V558Δ/+

mice (Sommer et al. 2003) was exacerbated in the KitV558Δ;T669I/+ mice (Fig. 5C and Fig.

S11G a and b). In approximately half the male KitV558Δ;T669I/+ mice we observed mast cell accumulation around Leydig cells in the interstitial space of the testes. Interestingly, KitV558Δ;T669I/+ mice exhibited intermittent partial alopecia of the trunk between postnatal day (P)15 and P40 (Fig. 5A), a phenotype reported for Il10^−/− mice and shown to be as-sociated with increased numbers of mast cells (Vanderford et al. 2010).

Importantly, the KitV558Δ;T669I/+ mice developed a pronounced microcytic erythro-cytosis. Manifestation of erythrocytosis first was detected phenotypically by reddening of the paws, distinguishable from wild-type littermates from P40 onwards (Fig. 5B).

Fig. 5: Increased mast cell and red blood cell numbers in KitV558Δ;T669I/+ mice.

(A) Representative photograph of alopecia of truncal hair in 4-wk-old KitV558Δ;T669I/+ mice. (B) Representa-tive photographs of wild-type hind-paw in old wild-type mice and “red paw” phenotype in 3-mo-old KitV558Δ;T669I/+ mice. (C) Increased mast cell numbers in dorsal skin of KitV558Δ;T669I/+ (GTK) mice in comparison with wild-type and Kit^V558Δ/+ mice (n = 3). (D) BM cellularity of KitV558Δ;T669I/+ mice measured as total nucleated cells per bone is similar to that in wild-type mice. Spleen cellularity is significantly in-creased in the mutants compared with wild-type mice. n = 6. (E) Time course of hematocrit and (F) MCV showing development of erythrocytosis and microcytosis phenotype in KitV558Δ;T669I/+ mice (red curves) and in wild-type mice (black curves). n = 5–17. Error bars indicate means ± SD.

Analysis of peripheral blood values of 8-wk-old animals revealed a substantial increase in erythrocyte counts and hematocrit values in comparison with wild-type and Kit^V558Δ/+

mice (Fig. S11A and B). Furthermore, the mean corpuscular volume (MCV) was de-creased significantly, and the mean platelet volume was inde-creased (Fig. S11C and D).

Scanning electron microscopy confirmed that erythrocytes were smaller in diameter in KitV558Δ;T669I/+ mice than wild-type mice but otherwise were morphologically normal (Fig. S11E and F). To gain insight into the kinetics of erythrocytosis and microcytosis development, we assessed hematocrit and MCV values in bleeding-naive (never before bled) KitV558Δ;T669I/+ mice at weeks 3, 4, 5, 6, 8, 10, 13, and 16. The hematocrit showed a steep increase between postnatal weeks 3 and 8 (Fig. 5E). In contrast, the MCV already was reduced in 3-wk-old animals and did not change significantly over time (Fig. 5F).

These results indicate that the development of the microcytosis is independent of the systemic erythrocyte overload (i.e., congestion of blood vessels in liver, spleen, and other organs), which becomes apparent only after 5 wk (Fig. S11G g and h). Of note, the microcytic erythrocytosis of the KitV558Δ;T669I/+ mice is the opposite phenotype of the macrocytic anemia associated with Kit and Kitl loss-of-function mutations (Russell 1979; Bernstein et al. 1990; Besmer 1997).

Cytokine Dependence and Pharmacological Inhibition of Erythroid Colony Growth in KitV558Δ;T669I/+

Mice.

In accordance with the elevated hematocrit values, spleens in the KitV558Δ;T669I/+

mice were enlarged compared with wild-type mice, and spleen cellularity of KitV558Δ;T669I/+

mice was increased 1.5-fold without gross alterations in splenic architecture (Fig. 5D and Fig. S11G c and d). Bone marrow (BM) cellularity and histology was unchanged (Fig. 5D and Fig. S11G e and f). Although the erythrocytosis was reminiscent of myeloproliferative neoplasms, we noted several marked differences: The platelet counts were not elevated in KitV558Δ;T669I/+ mice (Kit^+/+: 672 ± 248 × 10³/μL and KitV558Δ;T669I/+

681 ± 231 × 10³/μL), and there was no myelofibrosis in either the BM or spleen, even in 1-y-old KitV558Δ;T669I/+ mice.

KIT has important functions in erythroid progenitors that can be revealed by the for-mation of in vitro burst-forming unit erythroid (BFU-E) colonies in semisolid medium.

The number of BFU-Es obtained from the KitV558Δ;T669I/+ BM and spleen in the presence

of KitL, IL-3, and erythropoietin (EPO) was higher than in wild-type mice, and a con-comitant increase in cell-forming unit erythroid (CFU-E) numbers also was observed in the mutant BM and spleen (Tab. 4).

Tab. 4: Increased erythroid progenitors in spleen of KitV558Δ;T669I/+ mice.

BM Spleen

Colony type Kit^+/+ KitV558Δ;T669I/+ Kit^+/+ KitV558Δ;T669I/+

BFU-E 3,938 ± 1,042 6,386 ± 3,193 3,317 ± 1,436 16,198 ± 8,439 CFU-E 1,707 ± 910 4,562 ± 1,645 3,731 ± 1,759 25,575 ± 9,645*

BM and spleen cells from wild-type and KitV558Δ;T669I/+ mice were cultured in the presence of KitL (100 ng/mL), EPO (6 U/mL), and IL-3 (100 ng/mL). CFU-E and BFU-E colonies were scored at the end of 2 and 10 d, respectively. n =3 mice per group. *P < 0.05 in comparison with wild type.

KitL and EPO play a synergistic role in erythroid colony growth and survival (Nocka et al. 1990). To test whether wild-type and KitV558Δ;T669I/+

BFU-E formation differed in the presence of variable concentrations of cytokines, BM and spleen cells were cultured in the presence of decreasing concentrations of KitL and a fixed concentration (6 U/mL) of EPO (Fig. 6A and B). BFU-Es from KitV558Δ;T669I/+ mice showed significantly reduced dependence on KitL compared with those from wild-type mice. The hypersensitivity of KitV558Δ;T669I/+ erythroid colonies to KitL was emphasized by the observation that the BFU-Es from KitV558Δ;T669I/+ BM and spleen were phenotypically larger than wild-type colonies at each concentration of KitL tested (Fig. 6C). Furthermore, KitV558Δ;T669I/+

BFU-E growth was dependent on EPO, because no colonies were observed in presence of KitL alone without EPO (Fig. 6A and B). Importantly, EPO levels in the peripheral blood were not significantly different (Kit^+/+: 281.3 ± 195 pg/mL; KitV558Δ;T669I/+ 244.5.5

± 146 pg/mL) (Fig. 6D), indicating that the erythrocytosis in the KitV558Δ;T669I/+ mice was caused by increased KIT signaling in early erythroid progenitors.

To investigate the effect of pharmacologic intervention on the hematopoietic pheno-type in the KitV558Δ;T669I/+ mice, we analyzed erythroid progenitor numbers in the BM and the spleen of KitV558Δ;T669I/+ mice after 7 d of treatment with either imatinib or sunitinib. Imatinib treatment did not change the number of BFU-Es from BM and spleen compared with control vehicle-treated animals (Fig. 6E and F); in contrast, sunitinib treatment significantly reduced BFU-E numbers from both BM and spleen and concomitantly reduced CFU-E numbers from both organs (Fig. 6E and F), suggesting that, in addition to the ICC hyperplasia and GIST development, the erythrocytosis phe-notype of the KitV558Δ;T669I/+

mice is dependent largely on abnormal KIT kinase activity.

Fig. 6: KitV558Δ;T669I/+ erythroid progenitor growth is hypersensitive to KitL and is susceptible to sunitinib inhibition.

(A and B) BM (A) and spleen (B) cells were cultured in colony assays in the presence of EPO and de-creasing levels of KitL. KitV558Δ;T669I/+ BM gives rise to a significantly higher number of BFU-Es for each KitL+EPO combination. KitV558Δ;T669I/+ spleen also gives rise to significantly greater number of BFU-Es at all the KitL+EPO combinations tested compared with wild-type spleen. Error bars indicate means ± SD;

n = 3. (C) Representative images depict size of BFU-E colonies obtained from wild-type and KitV558Δ;T669I/+ BM. (Magnification: 10 ×.) (D) EPO levels from sera of wild-type and KitV558Δ;T669I/+ mice did not differ statistically. Error bars indicate means ± SD. (E and F) KitV558Δ;T669I/+ mice were treated with vehicle, imatinib, or sunitinib for 7 d. Femoral BM and spleen cells were cultured in the presence of KitL (100 ng/mL), EPO (6 U/mL), and IL3 (100 ng/mL). BFU-E (E) and CFU-E (F) colonies were scored after 10 d and 3 d, respectively. Error bars indicate means ± SD; n = 3.

Im Dokument Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse (Seite 31-43)