key: cord-0027013-ocbpfw6a
authors: Bosso, Giuseppe; Lanuza-Gracia, Pablo; Piñeiro-Hermida, Sergio; Yilmaz, Merve; Serrano, Rosa; Blasco, Maria A.
title: Early differential responses elicited by BRAF(V600E) in adult mouse models
date: 2022-02-10
journal: Cell Death Dis
DOI: 10.1038/s41419-022-04597-z
sha: e2b4e2908d60f2d456f005218cff7babc4a238d6
doc_id: 27013
cord_uid: ocbpfw6a

The BRAF gene is frequently mutated in cancer. The most common genetic mutation is a single nucleotide transition which gives rise to a constitutively active BRAF kinase (BRAF(V600E)) which in turn sustains continuous cell proliferation. The study of BRAF(V600E) murine models has been mainly focused on the role of BRAF(V600E) in tumor development but little is known on the early molecular impact of BRAF(V600E) expression in vivo. Here, we study the immediate effects of acute ubiquitous BRAF(V600E) activation in vivo. We find that BRAF(V600E) elicits a rapid DNA damage response in the liver, spleen, lungs but not in thyroids. This DNA damage response does not occur at telomeres and is accompanied by activation of the senescence marker p21(CIP1) only in lungs but not in liver or spleen. Moreover, in lungs, BRAF(V600E) provokes an acute inflammatory state with a tissue-specific recruitment of neutrophils in the alveolar parenchyma and macrophages in bronchi/bronchioles, as well as bronchial/bronchiolar epithelium transdifferentiation and development of adenomas. Furthermore, whereas in non-tumor alveolar type II (ATIIs) pneumocytes, acute BRAF(V600E) induction elicits rapid p53-independent p21(CIP1) activation, adenoma ATIIs express p53 without resulting in p21(CIP1) gene activation. Conversely, albeit in Club cells BRAF(V600E)-mediated proliferative cue is more exacerbated compared to that occurring in ATIIs, such oncogenic stimulus culminates with p21(CIP1)-mediated cell cycle arrest and apoptosis. Our findings indicate that acute BRAF(V600E) expression drives an immediate induction of DNA damage response in vivo. More importantly, it also results in rapid differential responses of cell cycle and senescence-associated proteins in lung epithelia, thus revealing the early molecular changes emerging in BRAF(V600E)-challenged cells during tumorigenesis in vivo.

The BRAF gene, encoding a master kinase of RAS-activated RAF-MEK-ERK (RAS-) pathway, is frequently mutated in human malignancies [1] [2] [3] [4] [5] [6] . More than 90% of these mutations affect codon 600 of the BRAF protein, and out of these,~90% represent the 1799T > A nucleotide transition, which results in a constitutively active [7] BRAF variant (BRAF V600E ) which indefinitely sustains cell proliferation.

The investigation of BRAF V600E genetically engineered mouse models (GEMMs) has been focused on the role of BRAF V600E in cancer in diverse tissues/organs [8] [9] [10] [11] [12] [13] [14] [15] . BRAF V600E expression in vivo triggers an early hyperplastic growth which culminates in a proliferative arrest known as oncogene-induced senescence (OIS) [9, 11, 15] , which is driven by p53/p21 CIP1 and retinoblastoma protein (Rb)/p16 INK4a pathways [16, 17] .

Albeit the above-mentioned GEMMs, where BRAF V600E expression relies on tissue-specific promoters, allowed to dissect the function of BRAF V600E in cancer, such an approach carries the limitation of lacking the global view of potential early effects induced by this oncogene. Indeed, apart from the initial wave of hyperplasia, the instant consequences on BRAF V600E -activation in vivo remain unexplored. Here we study the early events following acute expression of BRAF V600E in vivo.

To analyze the immediate impact of ubiquitous expression of BRAF V600E in vivo, we generated UbiCreER T2/+ ;BRAF LSL_V600E/+ (BRAF V600E ) mice harboring UbiCreER T2 allele [18] , expressing the conditionally active CreER T2 recombinase gene under the control of the human ubiquitin promoter (UbiCreER T2 ), combined with BRAF LSL_V600E allele [9] . First, we checked the viability of BRAF V600E mice in the absence of tamoxifen treatments. Albeit until the age of 9 weeks all the mice appeared healthy, starting from 10 weeks from birth they showed weight loss, locomotion alteration, bad shape, papillomatous skin lesions and all of them died between 12-18 weeks from birth ( Supplementary Fig. 1A, B) , a phenotype which is most likely due to the effects of a spontaneous Cremediated recombination of BRAF V600E allele over time. Consistently, PCR analysis of spontaneous papilloma-like lesions arisen in some of the tamoxifen-untreated BRAF V600E mice revealed Cremediated activation of BRAF V600E allele ( Supplementary Fig. 1C ).

To induce an acute BRAF V600E activation, 7-8 weeks old mice were administered tamoxifen intraperitoneally. The BRAF V600E mice, but not the UbiCreER T2/+ (control) strain, started to appear sick 2-3 days post-injection and needed to be euthanatized after 3-5 days (Supplementary Fig. 2A, B) . Macroscopic analysis revealed that BRAF V600E mice had pale livers, which may be indicative of hepatic steatosis (HS) (Supplementary Fig. 2C ). PCR analysis confirmed Cre-mediated rearrangement of BRAF V600E allele upon tamoxifen administration, resulting in BRAF V600E expression in all the tissues/organs analyzed ( Supplementary Fig.  2D ).

Early effects of BRAF V600E expression in thyroids, liver and spleen First, we analyzed the early effects of BRAF V600E expression in thyroids. Hematoxylin eosin (H&E) staining revealed no morphological alterations between BRAF V600E and control thyroids at 4-5 days after Cre-induction ( Supplementary Fig. 2E ). Phosphorylation analysis of the downstream effector ERK kinase (ppERK) revealed an increase in ppERK-positive cells in BRAF V600E thyroids compared to control, thus confirming that BRAF V600E is induced in thyroid glands and is stimulating the RAS-pathway ( Supplementary Fig. 2F ). However, BRAF V600E thyroids displayed no changes in apoptosis, as determined by caspase 3 (CC3), in senescence as determined by p21 CIP1 , or in DNA damage as indicated by the DNA damage protein γH2AX ( Supplementary Fig. 2G -I) compared to controls, indicating that acute BRAF V600E expression in thyroids does not have an apparent impact on cellular viability programs.

Next, we checked the immediate effects of BRAF V600E expression in the liver. In agreement with pale livers present in BRAF V600E mice at their end-point, H&E staining revealed the presence of microvesicular HS which was not present in age-matched controls ( Fig. 1A ) (see Discussion). BRAF V600E livers showed an enrichment in ppERK-positive cells, thus confirming RAS-pathway activation (Fig. 1B) . Concomitantly, albeit γH2AX-positive BRAF V600E hepatocytes were increased 2-fold compared to controls, neither CC3nor p21 CIP1 -positive cells were significantly altered ( Fig. 1C-E) , thus suggesting that after 3-5 days of acute expression, although BRAF V600E induces HS and DNA damage, it elicits neither apoptosis nor alterations in p21 CIP1 expression in the liver.

We next investigated the effects of BRAF V600E activation in the spleen. Histopathology analyses revealed a substantial loss of cellularity in both white and red pulp zones as well as the appearance of apoptotic bodies in BRAF V600E spleens but not in controls (Fig. 1F ). Only BRAF V600E spleens displayed loss of a clear boundary between the white and red pulp (Fig. 1F ), which is indicative of a general cell depletion [19] . Although we found ppERK activation in BRAF V600E spleens (Fig. 1G ), Ki67-positive proliferating BRAF V600E cells were significantly reduced (Supplementary Fig. 3 ), which is in agreement with the observed loss of cellularity. Coherently, CC3-positive cells were increased compared to controls, suggesting that acute BRAF V600E expression in the spleen results in lower proliferation and apoptosis induction (Fig.  1H) . Interestingly, γH2AX-positive cells in BRAF V600E spleens were increased by 7-fold compared to controls suggestive of enhanced DNA damage following BRAF V600E expression (Fig. 1I ). In contrast, we observed no accumulation of p21 CIP1 [20] (Fig. 1J) . Collectively, in the spleen acute BRAF V600E induction induces DNA damage without eliciting p21 CIP1 expression.

Early effects of BRAF V600E expression in lung alveolar parenchyma To investigate the immediate effects of BRAF V600E expression in lungs, we first confirmed that BRAF V600E was indeed expressed in lungs at the protein level by immunoblot analysis ( Supplementary  Fig. 4 ). Next, we found that BRAF V600E alveolar parenchyma showed alveolar wall thickening and adenomas ( Fig. 2A-C ). An increase in ppERK-and Ki67-positive cells confirmed that BRAF V600E leads to RAS-pathway activation and consequent proliferation in lungs (Fig. 2D, E) . We found no differences in alveolar CC3-positive cells between BRAF V600E and control mice (Fig. 2F) . Interestingly, BRAF V600E expression elicited a significant increment in γH2AX and p21 CIP1 protein levels, as well as in the number cells positive for such markers ( Supplementary Fig. 4, Fig.  2G , H). Moreover, telomere-induced foci (TIF) analysis revealed that BRAF V600E -elicited DNA damage is not telomeric (Supplementary Fig. 5A ). However, we found no changes in the frequency of cells expressing either p53, or p16 INK4a and p19 ARF , which are all expressed in senescent BRAF V600E -driven lung adenomas [21] (Fig.  2I-K) .

Altogether, these data suggest that in the alveolar parenchyma BRAF V600E elicits rapid DNA damage and p53-independent p21 CIP1 activation without inducing other classical hallmarks of senescence.

The finding that BRAF V600E mice displayed adenomas ( Fig.  2A -C) staining positive for the prosurfactant protein C (SPC), a marker of alveolar type II cells (ATIIs), and negative for CC10, a protein specifically expressed by Club cells (CCs), confirmed the evidence that BRAF V600E -induced adenomas display the properties of ATIIs [9, 17] (Fig. 3A, B) . Although the adenomas showed high levels of ppERK and Ki67, which are indicative of the BRAF V600E -triggered proliferative wave (Fig. 3C , D), they also showed increased DNA damage compared to controls (Fig. 3E) . Notably, as occurred in normal alveoli, even in the adenomas, BRAF V600E -induced DNA damage is not telomeric (Supplementary Fig. 5B ). Tumors also showed increased expression of CC3 and p21 CIP1 , whereas p53 + cells were incremented by 500-fold compared to control, thus suggesting apoptosis, p21 and p53 induction in the adenomas (Fig. 3F-H) . Moreover, we detected enriched protein levels of the senescent markers p15 INK4b , p16 INK4a and p27 KIP1 ( Supplementary Fig. 4 ), as well as in the occurrence of p16 INK4a -and p19 ARF -positive cells (Fig. 3I , J), therefore indicating that senescence is taking place in some of the adenoma cells. Collectively, these findings indicate that BRAF V600E -driven adenomas are composed by both proliferating and senescent cells.

Albeit in BRAF V600E mice both non-tumor alveolar parenchyma and adenomas showed increased p21 CIP1 levels compared to controls (Fig. 2H, Fig. 3G , Supplementary Fig. 4 ), neither smaller nor larger adenomas show significant changes in p21 CIP1 levels compared to non-tumor BRAF V600E alveolar zones ( Supplementary  Fig. 6A ). Nevertheless, only adenomas showed p53, p16 INK4a and p19 ARF upregulation ( Fig. 2I -K, Fig. 3H -J). Thus, we hypothesized that p21 CIP1 may be activated via p53 only in adenomas and that alternative mechanisms might be employed to induce p21 CIP1 in BRAF V600E -challenged non-tumor alveoli which might not reflect the activation of classical OIS. BRAF V600E is known to activate p21 CIP1 expression via E2F transcription factors in vitro, upon cyclin-dependent kinases (CDKs)-mediated Rb inactivation [22, 23] . Nevertheless, no alterations in the percentage of cells showing the inactivated phosphorylated version of Rb was observed either between BRAF V600E non-tumor areas and adenomas or between BRAF V600E and control mice ( Supplementary Fig. 6B ), thus suggesting that BRAF V600E -mediated p21 CIP1 activation in the alveolar parenchyma may not rely on pRb/E2F axis.

The RAS-pathway also induces small mother against decapentaplegic-3 (SMAD3) [24, 25] and STAT3 [26, 27] , two transcriptions factors whose phosphorylated versions (pSMAD3, pSTAT3) activate p21 CIP1 gene [28] [29] [30] [31] . Unexpectedly, we found no changes in the percentage of pSMAD3-positive cells among adenomas and non-tumor alveolar areas from both BRAF V600E mice and controls ( Supplementary Fig. 6C ). Conversely, both nontumor BRAF V600E parenchyma and adenomas showed increased pSTAT3 staining compared to controls ( Supplementary Fig. 6D ), thus indicating that BRAF V600E activates STAT3, but not SMAD3, in the lung and potentially suggesting that p21 CIP1 may be induced via STAT3 in BRAF V600E lung parenchyma. , J p19 ARF immunostainings in lung sections from BRAF V600E and control mice. Quantifications were performed on four different random areas of at least four hyperplastic nodules sections and four different random areas of uninduced alveolar parenchyma. Data are expressed as mean ± SEM; n represents respectively the number of animals per group in "normal epithelium" samples and the number of tumors in the "hyperplastic nodules" samples. For each condition at least 4 mice were used. *P < 0.05; **P < 0.01; ***P < 0.001, ns = not significant (T Student's test unpaired). Arrows point to selected positive cells for the indicated marker. Insets: magnifications of areas inside dashed squares.

To better analyze the differential response elicited by BRAF V600E in ATIIs, we checked whether BRAF V600E might affect the proliferation index of non-tumor and adenoma ATIIs. Double immunohistochemistry stainings with SPC and Ki67 markers revealed that upon BRAF V600E activation, albeit both non-tumor and tumor ATIIs from BRAF V600E mice tended to display a higher proliferation index, we found no significant changes in the number of SPC + Ki67 + cells (Fig. 4A) . Consistently, the percentage of ATIIs expressing pRb, another hallmark of cell cycle progression, among adenomas and non-tumor alveolar areas from both BRAF V600E and control mice was not affected (Fig. 4B) , therefore enforcing the evidence that acute BRAF V600E activation does not result in drastic changes in the expression of proliferative markers in ATIIs. Interestingly, both non-tumor and tumor BRAF V600E ATIIs displayed increased DNA damage (Fig. 4C) . Remarkably, only nontumor BRAF V600E ATIIs showed an increment in p21 CIP1 expression compared to both controls and adenoma ATIIs (Fig. 4D) , thus indicating that the majority of p21 CIP1 -positive cells in adenomas are not SPC + ATIIs and that the oncogenic challenge elicits a rapid p21 CIP1 activation in ATIIs before they can give rise to adenomas ( Supplementary Fig. 6A) . Surprisingly, whereas there was no alteration in p53 expression in non-tumor BRAF V600E ATIIs, we observed a robust p53 induction in adenomas (Fig. 4E ), thus suggesting that p21 CIP1 activation in non-tumor ATIIs does not rely on p53 and that p53 induction in adenoma ATIIs does not promptly result in p21 CIP1 expression. Moreover, double staining experiments by using SPC marker combined with either pSMAD3 (Supplementary Fig. 7 ) or pSTAT3 (Fig. 4F ) showed a drastic increment in pSTAT3, but not in pSMAD3, in BRAF V600E mice compared to control, thus confirming that 1) the BRAF V600Edependent increase of pSTAT3 in the lungs (Supplementary Fig.  6D ) is ascribable to pSTAT3 enrichment in ATIIs and 2) arguing for the possibility that pSTAT3 activation may be uncoupled from cell proliferation in BRAF V600E -challenged ATIIs, at least at the specific stages of oncogene-induced cell transformation analyzed (Fig. 4A , B, F). Altogether, these findings indicate that albeit BRAF V600E induces DNA damage as well as pSTAT3 in ATIIs outside and inside the adenomas, it results in differential p21 CIP1 and p53 expression in non-tumor and adenoma ATIIs.

Early effects of BRAF V600E in bronchial/bronchiolar epithelium and Club cells We next studied the early effects of BRAF V600E expression in bronchi/bronchioles. Following BRAF V600E activation, bronchial/ bronchiolar cells showed a significant loss of cells positive for the specific CC marker CC10 (Club cell secretory protein 10KDa) (Fig.  5A) . Furthermore, intensity of CC10 staining was also significantly reduced ( Supplementary Fig. 8A ). Concomitantly, SPC + intrabronchial cells were drastically increased (Fig. 5B) , thus suggesting that BRAF V600E induces transdifferentiation of CCs into ATIIs. Moreover, an increment in Ki67-positive cells (Fig. 5C) was accompanied by respectively a 2-fold and 8-fold increase in the intensity of cyclin D1 staining and in the number of pRb-positive cells in BRAF V600E bronchi/bronchioles compared to controls (Fig. 5D, E) , therefore indicating a dramatic stimulation of cell cycle progression. Simultaneously, BRAF V600E mice displayed a 2-fold increase of γH2AX-positive cells (Fig. 5F ) and although they tended to display more p53 + cells, such an increase did not reach statistical significance. Nevertheless, p21 CIP1 -and CC3-positive bronchial/ bronchiolar BRAF V600E cells were enriched by 15-fold and 2-fold respectively compared to controls (Fig. 5H, I) , thus suggesting that the dramatic proliferative cues observed upon BRAF V600E challenge culminates in a robust p53-independent p21-mediated cell cycle arrest and cell death. Intriguingly, there were no changes in the frequency of p16 INK4a -and p19 ARF -positive cells (Fig. 5J, K) , thus suggesting that p21 CIP1 increase is not coupled to other bronchi/bronchioles senescence markers [32] .

To investigate the early BRAF V600E -driven responses in CCs, we first checked the proliferation index of BRAF V600E CCs. Contrary to ATIIs, double immunohistochemistry staining with CC10 and Ki67 markers revealed that BRAF V600E CCs showed a dramatic 20-fold and 15-fold increase respectively in Ki67 and pRb (Fig. 6A, B) , thus confirming the finding that CCs are sensitive to BRAF V600E -mediated proliferation stimulation. However, additional double immunostainings with CC10, γH2AX and p21 CIP1 markers revealed that BRAF V600E CCs showed respectively a 5.4-fold and 10-fold increase in γH2AXand p21-positive cells compared to controls (Fig. 6C, D) , therefore enforcing the evidence that BRAF V600E also elicits a dramatic induction of DNA damage and p21 CIP1 in CCs.

Interestingly, we found the same expression pattern described above also in SPC + CCs. Indeed, transdifferentiating BRAF V600E CCs were positive for Ki67 and pRb (Fig. 6E, F) , as well as for γH2AX and p21 CIP1 markers (Fig. 6G, H) , thus indicating that the BRAF V600Emediated proliferation stimulation coexists with the cytotoxic response during the early steps of CC-to-ATII transdifferentiation. It is worth pointing out that bronchial/bronchiolar cells staining positive or negative for SPC show the same frequency in p21 CIP1 -, γH2AX-, Ki67-, pRb-positive cells ( Supplementary Fig. 8B-E) , thus suggesting that the increment respectively in p21 CIP1 expression, DNA damage and proliferation may not affect the onset of the transdifferentiation process.

Altogether, these findings unveil that upon BRAF V600E -challenge CCs transdifferentiate and massively activate a robust cell cycle progression signaling which rapidly culminates in cell cycle inhibition and apoptosis. p21 CIP1 activation in lungs is the consequence of acute tamoxifen-mediated BRAF V600E induction Next, we ruled out the possibility that p53-independent p21 CIP1 activation in bronchi/bronchioles and in non-tumor alveolar parenchyma might be ascribable to a prolonged effect of BRAF V600E chronic activation, which may be spontaneously occurred at some earlier time-points before tamoxifen injections. For this purpose, concomitantly with tamoxifen-treated BRAF V600E mice, we also analyzed the lungs of 10-11 weeks old BRAF V600E mice without previous tamoxifen treatment. Remarkably, although such untreated mice showed spontaneous lung adenomas expressing high levels of p21 CIP1 and p53, we found no differences in both these proteins in non-tumor alveolar parenchyma of BRAF V600E mice compared to untreated age-matched controls ( Supplementary Fig. 9A, B) . Similarly, we observed no change in either Ki67 or p21 CIP1 in bronchi/bronchioles compared to controls ( Supplementary Fig. 9C, D) , thus enforcing the evidence that the proliferation induction and p21 CIP1 activation in non-tumor alveolar parenchyma and in bronchi/bronchioles observed in tamoxifen-treated BRAF V600E mice is an immediate and acute effect of BRAF V600E expression rather than a cumulative effect over time of random events of Cre-dependent recombination of Fig. 4 BRAF V600E induction results in differential expression of cell cycle and senescence markers in ATII cells from uninduced, induced non-tumor alveolar parenchyma and lung adenomas. Representative images (from the left) and quantifications (right) showing SPC+ cells staining positive for A Ki67, B pRb, C γH2AX, D p21 CIP1 , E p53 and F pSTAT3 double immunostainings in normal alveolar epithelium of control mice (left), non-tumor areas (center) and adenomas (right) from lung sections of BRAF V600E mice. Quantifications were performed on four to ten different random areas of at least four hyperplastic nodules sections and four to ten different random areas of uninduced alveolar parenchyma. Data are expressed as mean ± SEM; n represents respectively the number of animals per group in "alveoli" samples and the number of adenomas in the "tumor" samples. For each condition at least 4 mice were used. The percentages shown in the charts were obtained dividing the number of double positive cells by the overall number of SPC + cells. *P < 0.05; **P < 0.01; ***P < 0.001, ns = not significant. (ANOVA test with Tukey's post-hoc correction). Arrows point to selected positive cells for the indicated marker. BRAF V600E allele which may lastly result in a basal/chronic BRAF V600E -activation.

We also checked whether BRAF V600E might elicit acute lung inflammation. Interestingly, 4 days after BRAF V600E -induction, we found a dramatic infiltration of neutrophils, harboring the characteristic multilobed nuclei and staining positive for the neutrophilic marker myeloperoxidase (MPO) (Supplementary Fig.  10A, B) in the alveolar parenchyma of BRAF V600E mice but not in controls. Nevertheless, we found no enrichment of cells positive for the monocyte/macrophagic marker F4/80 in the alveoli (Supplementary Fig. 10C) . Furthermore, the occurrence of CD4 + T-lymphocytes in BRAF V600E mice was reduced twice compared to control ( Supplementary Fig. 10D ), thus indicating that BRAF V600E ubiquitous expression induces an immediate alveolar infiltration of neutrophils as well as a loss in CD4 + lymphocytes without affecting monocytic/macrophagic lineage.

Conversely, albeit F4/80 + cells were increased in BRAF V600E bronchi/bronchioles compared to controls (Supplementary Fig.  10E ), neither neutrophils nor CD4 + lymphocytes frequencies were perturbed (Supplementary Fig. 10F, G) , thus suggesting that BRAF V600E triggers an immediate bronchial infiltration specifically of F4/80 + cells. Similarly, also adenomas displayed a specific enrichment in F4/80 + cells ( Supplementary Fig. 10H ), but not in either MPO-or CD4-positive cells ( Supplementary Fig. 10I, J) , thus possibly indicating that BRAF V600E -driven adenomas may preferentially recruit macrophages rather than neutrophils or lymphocytes.

Furthermore, double immunostainings revealed that the percentages of F4/80 + cells positive for the anti-inflammatory M2 macrophagic markers pSTAT3 [33] [34] [35] peroxisome-activated proliferator receptor-γ (PPARγ) [33, 36, 37] and c-MYC [33, 38, 39] were globally enhanced in both bronchial/bronchiolar and alveolar parenchyma of mutant mice as well as in adenomas compared to control (Fig. 7A-F) , thus indicating a BRAF V600Emediated overall increase in pro-tumoral macrophages in lungs. Conversely, F4/80 + cells positive for the pro-inflammatory M1 macrophagic marker hypoxia inducible factor-1α (HIF1α) [35, [40] [41] [42] were significantly increased only in adenomas, but not in either alveolar or bronchial/bronchiolar parenchyma of mutant mice compared to controls ( Supplementary Fig. 11A, B) , thus giving further confirmation on the ability of BRAF V600E to orchestrate the immediate recruitment of specific leukocytes in different pulmonary epithelia.

We finally checked whether BRAF V600E might result in reactive oxygen species (ROS) production in vivo. Interestingly, immunostaining experiments revealed increased levels of the ROS markers 4-hydroxy-2-nonenal [43] [44] [45] [46] and 8-hydroxy-2'-deoxyguanosine [43, [47] [48] [49] [50] in the spleen but not in lungs, liver, or thyroids of BRAF V600E -mice compared to controls, thus suggesting that, albeit the BRAF V600E -induced DNA damage may be ROS-dependent in vivo as well as in vitro [51] , additional mechanisms might be involved in BRAF V600E -dependent DNA damage induction in lungs and liver (Fig. 8A-H) .

Here we reported that ubiquitous acute BRAF V600E expression leads to a rapidly lethal sickness characterized by general weakness and weight loss. This outcome may be partially ascribable to lung acute inflammation and to a rapid energetic depletion likely attributable to BRAF V600E -triggered lipolysis, a process which is mediated by activated RAS-pathway [52] . We also observed that BRAF V600E activation induces microvesicular HS, a condition where an excess of fatty acids is accumulated into hepatocytes. Thus, it is conceivable that the BRAF V600E -triggered hepatic fat accumulation may be ascribable to a likely increase of serum fatty acids consequent to the RAS-pathway-driven lipolysis in adipocytes [52] .

Our findings provided the first evidence in vivo that acute BRAF V600E expression elicits instant DNA damage in an organspecific fashion. p21 CIP1 [53] , which may be activated by p53 upon genotoxic insults [54] and by oncogene activation via pRb/E2F [22] , promotes cell cycle arrest and senescence [22] by inhibiting CDKs [22] . Nevertheless, despite BRAF V600E induces both DNA damage and p21 CIP1 activation in vitro [51, 55] as well as in senescent lung adenomas [17] , we found no differences in p21 CIP1 levels either in liver or spleen upon BRAF V600E expression. Thus, we unveiled that, in the organs where BRAF V600E rapidly induces robust DNA damage, an immediate p21 CIP1 activation does not occur in a generalized manner. Such observations suggest that p21 CIP1 may be activated only at later time points in the presence of a constant oncogenic stimulus, or that BRAF V600E ability to induce DNA damage in certain tissues/organs may be uncoupled from p21 CIP1 activation.

We also uncovered that BRAF V600E expression yields a differential response of cell cycle/senescence-associated proteins in ATIIs. Indeed, albeit all the BRAF V600E -challenged ATIIs showed increased DNA damage, while non-transformed ATIIs express p21 CIP1 in the absence of p53 activation, tumorigenic ATIIs displayed enhanced p53 expression coupled with a significant p21 CIP1 reduction compared to non-tumor cells. The strike differences in such expression patterns argue for the possibility that non-tumor ATIIs may represent an early stage of tumor development in which a rapid p53-independent-p21 CIP1 induction might be an immediate barrier to cancer initiation which, at a certain point, may be repressed thus allowing cell proliferation. Alternatively, some ATIIs might be naturally more refractory to an immediate BRAF V600E -dependent p21 CIP1 activation and be more prone to give rise to adenomas, which lastly, during the onset of senescence, will activate p53. Thus, the evidence that p53 induction in adenoma ATIIs is accompanied by no alteration in p21 expression might be due to the fact the p53 activation is at an initial stage and therefore it has not reached yet the threshold necessary for an efficient p21 CIP1 gene activation.

Albeit it has been established that BRAF V600E promotes senescence or apoptosis without yielding any previous proliferative stimulation in vitro [51, 56] , BRAF V600E expression in vivo results in an initial hyperplasic wave lastly culminating in the onset of tumor senescence, characterized by DNA damage [17] , and p21 CIP1 [17] , p19 ARF [9] , p16 INK4a [21] and p53 [57] expression. Nevertheless, we unveiled that in BRAF V600E mice, non-tumor alveolar parenchyma showed rapid p21 CIP1 induction, which is not accompanied by activation of any among the well-known proteins associated to OIS in lungs [9, 17] , thus arguing for the possibility that such immediate tumorsuppression response may differ from classical OIS. Indeed, such p53/pRb-independent p21 CIP1 activation in non-tumor alveolar parenchyma may reflect a rapid BRAF V600E -mediated cytotoxic response reminiscent to that observed in CCs (see below), thus suggesting that albeit BRAF V600E ATIIs are prone to give rise to adenomas, yet there is an immediate cell cycle arrest in a minority of the challenged ATIIs.

In contrast to the extensive research work conducted in ATIIs, very little is known about the early molecular effects of BRAF V600E in bronchi/bronchioles. Here we unveiled that BRAF V600E initiates CCs transdifferentiation into ATIIs. Concomitantly, we observed a proliferation stimulation resulting in DNA damage, cell cycle arrest and cell death. Both proliferative and cytotoxic responses are much more exacerbated in CCs compared to ATIIs, which can account for the well-known characteristic of CCs to be recalcitrant to RAS-pathway stimulation [58, 59] .

We also uncovered that BRAF V600E rapidly elicits an acute inflammatory response in lungs by differentially recruiting neutrophils in the alveoli and F4/80-positive cells in bronchi/ bronchioles and adenomas. The generation of GEMMs in which BRAF V600E expression is driven specifically in neutrophils and alveolar macrophages will provide helpful insights into the mechanisms underlying the BRAF V600E pleiotropic effect on leukocytes in lungs.

Murine models BRAF LSLV600E mice were described previously [9, 10, 12] . This mouse model was crossed with a mouse strain carrying ubiquitously expressed, tamoxifen-activated recombinase, UBC-CreER T2 [18] , to generate UBC-CreER T2/+ ;BRAF LSL_V600E/+ mice. These mice received intraperitoneal injections of 4-hydroxy tamoxifen (Sigma H6278) (1 mg/injection, 3-4 injections, 1 injection per day for 3 or 4 consecutive days).

All mice were maintained at the Spanish National Cancer Research Centre under specific pathogen-free conditions in accordance with the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). All animal experiments were approved by our Institutional Animal Care and Use Committee (IACUC) and by the Ethical Committee for animal experimentation (CEIyBA) (PROEX 106.7/20). We followed the Reporting in Vivo Experiments (ARRIVE) guidelines developed by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs). Both male and female mice, with mixed background, were used for the experiments.

Immuno-FISH was performed in formalin-fixed paraffin-embedded mouse lung sections to identify telomeric induced foci (TIF) as previously described [60, 61] . Immuno-FISH was performed as follows: after deparaffination and citrate antigen retrieval, samples were permeabilized for 3 h in PBS1X-0.5% Triton, blocked for 2 h with 10% fetal bovine serum and 1 h with 5% BSA in PBS1X-0.1%Triton-10mM Glycine (PBSTG), and immunofluorescence with anti-53BP1 rabbit antibody (Novus Biologicals NB100-304) diluted 1:500 was performed. Samples were incubated O/N at 4°C with the primary antibody in PBSTG. Slides were further washed with PBSTG and incubated with 488-Alexa labeled secondary antibody in DAKO antibody diluent reagent (S3022). After immunofluorescence, samples were fixed for 20 min in 4% paraformaldehyde in PBS1X and followed by FISH. Briefly, samples were washed with PBS and dehydrated in Ethanol 70, 90 and 100%. The samples were then incubated with a telomeric PNA probe labeled with CY3 (Panagene) in 50% formamide for 30 min, washed in the presence of 50% formamide and counterstained with DAPI. TIF were identified by colocalization of CY3 and 488-Alexa double positive spots. Confocal microscopy was performed at room temperature with a laser-scanning microscope (TCS SP5; Leica) using a Plan Apo 63Å-1.40 NA oil immersion objective (HCX; Leica). Maximal projection of Z-stack images generated using advanced fluorescence software (LAS) was analyzed with the Definiens XD software package. The DAPI images were used to detect signals inside the nuclei.

Tissues were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For histological examination sections were stained with hematoxylin and eosin, according to standard procedures as previously described [62] . 

Protein extracts were obtained as follows: 45 mg of lung for each mouse were mechanically homogenized in 850ul lysis buffer (50 mM TrisHCl pH 7.5, 420 mM NaCl, 1% Triton, 1 mM EDTA, 2.5 mM MgCl2, protease inhibitors) in BERTIN Precellys 24 Lysis & Homogenization machine, incubated 30 min on ice in agitation, sonicated 10 sec, centrifuged at 14000 g for 20 min at 4°C. The recovered supernatant was passed through a 0.22 filter, aliquoted, flash-frozen in liquid nitrogen and stored at −80°C. Protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad). 40 µg of nuclear protein extracts were separated in SDS-polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane, the membranes were incubated with the indicated antibodies: monoclonal anti-actin 1:5000 (A5441, Sigma), anti-BRAF 1:200 (F-7, sc-5284, Santa Cruz), anti-BRAF V600E 1:300 (31-1042-00 RevMAB Biosciences USA), anti-γH2AX Ser139 1:5000 (Merck 05-636), homemade rat anti-p15 INK4b clone PAT65B (neat supernatant), homemade rat anti-p16 INK4a clone PABLO33B (neat supernatant), handmade rat anti-p19 ARF clone PIL346C (neat supernatant), homemade rat anti-p27 KIP1 clone SON82D (neat supernatant), homemade rat anti-p21 CIP1 clone HUGO291 (neat supernatant), homemade rat anti-p53 clone POE316A (neat supernatant). Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare) with Chemidoc (Biorad). For the quantification, protein-band intensities were quantified by densitometric analysis with ImageLab software (Biorad). The total levels of each protein analyzed have been normalized versus actin and the mean of the specific protein/actin ratio deriving from at least 3 different replicates has been used to generate the chart as previously described [63] . PCR DNA of tissue samples was extracted using Phenol:Chloroform:Isoamyl: Alcohol (Sigma). We determined Cre-mediated recombination by using the following PCR program: 94°C for 3 min, followed by 33 cycles of 94°C denaturation for 25 s, 25 s annealing at 55°C, elongation at 73°C for 45 s, followed by a 4 min 73°C elongation step with the following primes: Fw 5'-TGAGTATTTTTGTGGCAACTGC and Rev 5'-CTCTGCTGGGAAAGCGGC. This oligonucleotide primer pair hybridizes in intron 14 flanking the cassette insertion site. These conditions produce diagnostic PCR products of 185 bp for the wild-type BRAF and 308 bp for BRAF V600E alleles and a 335 bp PCR product for the Cre-activated BRAF V600E allele. The samples were resolved in a 3% agarose gel.

Immunohistochemistry quantifications were performed by direct cell counting by using Zen3.1 Zeiss and Image J softwares. ImmunoFISH quantifications were carried out by direct counting of cells and 53BP1 foci on single plans of each z-stack by using LAS X software (Leica). Unpaired Student's t-test (two-tailed), ANOVA followed by Tukey's post-hoc correction, Log Rank test were used to determine statistical significance. P-values of less than 0.05 were considered significant. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was performed using Microsoft® Excel 2016 and GraphPad/PRISM8. For animal studies no blinding/randomization was done/used. The number of mice per each experiment as well as the size of the experiments were obtained by performing power analysis.

The datasets and other information that support the findings of this study are available from the corresponding author upon reasonable request.

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We thank the Comparative Pathology and Mouse Facility Units at CNIO. MAB laboratory is funded by Spanish State Research Agency (AEI), Ministry of Science and Innovation, cofunded by the European Regional Development Fund (ERDF) (SAF2017-82623-R and SAF2015-72455-EXP), the Comunidad de Madrid Project (S2017/BMD-3770), the World Cancer Research (WCR) Project , the European Research Council (ERC-AvG Shelterines GA882385) and the Fundación Botín (Spain). GB is a Juan de la Cierva Incorporación post-doctoral fellow. MY is a FEBS PhD fellow.

MAB conceived the idea. MAB and GB designed the experiments. GB, PL, SPH and MY performed the experiments. MAB and GB wrote the manuscript. RS aided with mice treatments.

The authors declare no competing interests.

All authors approved and directly participated in the planning and/or execution of the experiments and/or analysis of the data presented herein. The animal studies were conducted in accordance with the Animal Use Protocol approved by theInstitutional Animal Care and Use Committee (IACUC) and by the Ethical Committee for animal experimentation (CEIyBA) (PROEX 106.7/20).

All authors have provided their consent for publication.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41419-022-04597-z.Correspondence and requests for materials should be addressed to Maria A. Blasco.Reprints and permission information is available at http://www.nature.com/ reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.