Fig 2: Dependency on Myc is Rapidly Acquired by Lung Adenocarcinomas(A) Representative immunostaining for Ki67, CD206, TUNEL (white arrows), and CD31 in adenocarcinomas at indicated time points following Myc activation for 7 days (7d tam) and subsequent de-activation (7d tam, then 7d off) compared to KRasG12D-only (control). The tumor edges boxed in the top panels are shown enlarged below. Scale bars apply across each row.(B) Quantification of histological changes in Ki67, CD206, TUNEL, CD3, and NKp46 in lung tumors after short term (7d tam) Myc-activation and subsequent Myc de-activation for 3 and 7 days (3d, 7d off) compared to KRasG12D-only control (7d oil).(C) Fold change in tumor cell proliferation (Ki67) and death (TUNEL) in mice treated with oil (control) or with tamoxifen for 7 days followed by tamoxifen withdrawal for 3 or 7 days. Individual and average values of tumor cell death and proliferation are displayed in (B).(D) Representative H&E staining of part of and whole lung lobes together with corresponding quantification of total tumor burden (left y axis) and tumor multiplicity (right y axis) in lungs of mice treated with tamoxifen for 7 days followed by Myc de-activation for either 7 days or 4 weeks (7d, 4w off) and compared to KRasG12D-only control (7d oil). T = tumor.Quantification graphs: FoV = field of view. (B): n = 25 individual tumors (small symbols) from 5 mice (large symbols) per time point. (D) Each individual data point represents a single mouse (n = 8 mice per group). Box and whisker graphs represent tumor multiplicity of 8 mice per group. Error bars represent the median with interquartile range (Ki67, CD206, TUNEL, CD3) or mean ± SD (NKp46). p values are based on Student’s t test (Ki67, CD206, TUNEL, CD3) or two-way ANOVA (NKp46). NS = non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.See also Figure S1.

Fig 3: Schematic Representations of Animal Experiments, Related to Figures 1, 2, 3, 4, 5, 6, and 7(A) Related to Figures 1 and S2. Analysis of long-term co-operation between KRasG12D and MycERT2. I, II and III denote three different regimens, each with a different time points of activation of MycERT2 (0, 6 and 12 weeks) post-AdV-Cre activation of KRasG12D.(B) Related to Figures 2, 4, and S3. Analysis of short-term MycERT2 activation in KRasG12D-driven KM mouse lungs. 12 weeks after AdV-Cre activation of KRasG12D in KM mice, MycERT2 was co-activated for 1, 3 or 7 days.(C) Related to Figures 3 and S4. Analysis of the impact of individual or co-blocking CCL9 and/or IL23p19 on MycERT2-driven KM lung tumor progression. 14 weeks after AdV-Cre treatment, and commencing one day prior to tamoxifen injection, mice were injected every other day for 4 days with neutralizing antibodies against either IL23p19 or CCL9 or both IL23p19 and CCL9, then euthanized.(D) Related to Figures 3 and S4. Analysis of the impact of long-term co-blocking CCL9 and/or IL23p19 on MycERT2-driven KM lung tumor progression. 12 weeks after AdV-Cre treatment, and commencing one day prior to tamoxifen injection, mice were injected every other day for 7 days with neutralizing antibodies against both IL23p19 and CCL9, then euthanized.(E) Related to Figure 4. Analysis of the impact of PD-L1 antibody blockade on MycERT2-driven KM lung tumor progression. 12 weeks after AdV-Cre administration, and commencing one day prior to tamoxifen injection to activate MycERT2, mice were injected every two days for two weeks with either PD-L1 neutralizing antibodies or IgG control.(F) Related to Figures 5 and S5. Analysis of short-term MycERT2 de-activation in KM lung tumors. 6 weeks after AdV-Cre activation of KRasG12D in KM mice, MycERT2 was co-activated for 6 weeks and then de-activated for 0, 1, 3 and 7 days.(G) Related to Figure 6. Analysis of the impact of short-term activation and short-term de-activation of MycERT2 in KRasG12D+Myc-driven KM lung tumors. 12 weeks after AdV-Cre activation of KRasG12D in KM mice, MycERT2 was co-activated for 1 week and then de-activated for 3 or 7 days.(H) Related to Figure 6. Analysis of the impact of short-term activation followed by long-term de-activation of MycERT2 in KRasG12D+Myc-driven KM lung tumors. 12 weeks after AdV-Cre activation of KRasG12D in KM mice, MycERT2 was co-activated for 1 week and then de-activated for up to 4 weeks.(I) Related to Figures 7 and S6. Analysis of the absence of T or NK cells during short-term de-activation of MycERT2 in KRasG12D+Myc-driven KM lung tumors. 12 weeks after AdV-Cre treatment, and starting four days prior to tamoxifen injection, KM mice were injected every other day for four cycles with neutralizing antibodies against CD8 (or IgG control) and every four days for two cycles (at -4 and 0 day time points) with neutralizing antibodies against CD4 (or IgG control), or every other day for four cycles with neutralizing antibodies against NKp46 (anti-asialo-GM1) then sacrificed one day after the last antibody injection coincident with day 3 post MycERT2.

Fig 4: Depletion of NKp46+ Cells, but Not of CD4+ and CD8+ T Cells, Retards Reversion of Myc-Driven Tumor Stromal Changes following Myc De-activation, Related to Figure 7(A) Quantification of immunostaining for proliferation (Ki67), CD206+ macrophages and NK cells (NKp46) following Myc de-activation for 3 days in KM tumors of either CD4+/CD8+ T cell competent animals (IgG-control) or CD4+/8+ T cell-deficient (aCD4/aCD8-treated) mice compared to 7 days of Myc activation (7d tam). No significant differences in IgG control versus T cell depleted animals are evident.(B) Quantification of immunostaining for proliferation (Ki67), CD206+ macrophages and T cells (CD3) following Myc de-activation for 3 days in KM tumors of either NKp46+ NK cell competent animals (IgG-control) or NKp46+ NK cell-deficient (a-asialoGM1-treated) mice. No significant differences in IgG control versus NK-cell depleted animals are evident for proliferation (Ki67) or T cells (CD3). In contrast, NK-cell depleted animals show reduced CD206 efflux following Myc de-activation.Quantification graphs: FoV = Field of View. (A): n = 20-25 individual tumors (small symbols) from 4 (7d Tam, IgG, large symbols) or 5 (aCD4/aCD8) mice per treatment group. (B): n = 25-30 individual tumors (small symbols) from 5 mice (large symbols) per treatment group. Error bars represent the median with interquartile range (Ki67, CD206, CD3) or mean ± SD (NKp46). P values are based on Student’s t test (Ki67, CD206, CD3) or two-way ANOVA (NKp46). NS = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: IL-23 and CCL9 Mediate Myc-Induced Remodeling of Lung Tumor Stroma(A) Representative inflammation antibody arrays probed with whole-lung protein lysates from mice treated for 8 hr with oil (control) or tamoxifen to activate Myc.(B) Quantification of IL12p40/70 and CCL9 signals derived from arrays shown in (A). Each individual data point represents a single mouse.(C) Quantification and representative examples of immunostaining for CD206, CD3, CD31, NKp46, B220, Ki67, and TUNEL after Myc activation for 3 days in mice sham treated (IgG control), treated with either IL23p19- or CCL9-blocking antibodies, or co-treated with both IL23p19- and CCL9-blocking antibodies. Boxed areas in each image are shown enlarged in the panels directly below. Scale bars apply across each row.(D) Quantification of tumor cell proliferation (Ki67, left) and cell death (TUNEL, right) after Myc activation for 7 days (with tamoxifen) in mice co-treated with either IgG control or co-treated with IL23p19- and CCL9-blocking antibodies.(E) Quantification of fold change in tumor cell death (TUNEL) and proliferation (Ki67) (left) and tumor burden (right) after Myc activation for 7 days (with tamoxifen) in mice co-treated with either IgG control or co-treated with IL23p19- and CCL9-blocking antibodies.Quantification graphs: FoV = field of view. Small symbols = individual tumors, large symbols = average per mouse. (C) n = 4 mice and n = 25–35 tumors (individual anti-IL23p19 or anti-CCL9 treatment) or n = 5 mice and n = 30–50 tumors (IgG control or anti-IL23p19 and anti-CCL9 co-treated). (D) n = 30 tumors from 6 mice treatment point. (E) n = 6 mice per treatment group. Error bars represent the median with interquartile range. p values are based on Student’s t test (CD206, CD3, CD31, B220, Ki67, TUNEL) or two-way ANOVA (NKp46). NS = non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.See also Figures S1, S4, and S7.