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# Deregulation of the p19/CDK4/CDK6 axis in Jak2V617F megakaryocytes accelerates the development of myelofibrosis

Hélène Duparc, Delphine Muller, Laure Gilles, Agathe Chédeville, Mira El Khoury, Rose Guignard, Najet Debili, Monika Wittner, Alexandre Kauskot, Florence Pasquier, et al.

► To cite this version:

Hélène Duparc, Delphine Muller, Laure Gilles, Agathe Chédeville, Mira El Khoury, et al. Deregulation of the p19/CDK4/CDK6 axis in Jak2V617F megakaryocytes accelerates the development of myelofibrosis. Leukemia, 2024, 38 (4), pp.898-902. 10.1038/s41375-024-02170-5. hal-04785472

HAL Id: hal-04785472

https://hal.science/hal-04785472v1

Submitted on 15 Nov 2024

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1 Deregulation of the p19/CDK4/CDK6 axis in Jak2<sup>V617F</sup> megakaryocytes accelerates the
development of myelofibrosis

3 Duparc Hélène<sup>1,2,3,4</sup>, Muller Delphine<sup>1,2,3*</sup>, Gilles Laure<sup>1,2,3*</sup>, Chédeville Agathe L<sup>1,2,3,4</sup>, El

4 Khoury Mira<sup>5</sup>, Guignard Rose<sup>1,2,3</sup>, Debili Najet<sup>1,2,3</sup>, Wittner Monika<sup>1,2,3</sup>, Kauskot Alexandre<sup>6</sup>,

5 Florence Pasquier<sup>1,2,7</sup>, Antony-Debré Iléana<sup>1,2,3</sup>, Marty Caroline<sup>1,2,3</sup>, Vainchenker William<sup>1,2,3</sup>,

6 Plo Isabelle<sup>1,2,3</sup>, Raslova Hana<sup>1,2,3</sup>

7

8 <sup>1</sup>INSERM, UMR1287, Gustave Roussy, Villejuif, France, Equipe labellisée Ligue Nationale

9 Contre le Cancer

10 <sup>2</sup> Université Paris-Saclay, Gif-sur-Yvette, Villejuif, France

11 <sup>3</sup> Gustave Roussy, Villejuif, France

12 <sup>4</sup> Université Paris Cité, Paris, France

13 <sup>5</sup>INSERM UMRS 938, PARIS, Sorbonne Université, Centre de Recherche Saint- Antoine,

14 AP-HP, Hôpital Saint-Antoine, Paris, France

15 <sup>6</sup>INSERM U1176, Hemostasis, Inflammation & Thrombosis (HITh), Université Paris-Saclay,

16 Le Kremlin-Bicêtre, France.

17 <sup>7</sup> Département d'Hématologie, Gustave Roussy, Université Paris-Saclay, Villejuif, France

18

19

20 *Equal contribution

21 **Corresponding author:** Hana RASLOVA, INSERM UMR1287, Gustave Roussy, Université

22 Paris-Saclay, 114 rue Edouard Vaillant, 94805 Villejuif, France; Phone: (+33) 1 42 11; Fax:

23 (+33) 1 42 11 52 40; E-mail: hana.raslova@gustaveroussy.fr

24 **Disclosure of Conflict of Interest:** The authors declare that there are no conflicts of interest.

25 **Running title:** "Palbociclib treatment of myelofibrosis"

26
27 Primary and secondary myelofibrosis (MF) present the worst prognosis in the group of 28 BCR::ABL-negative myeloproliferative neoplasms and are driven in half of cases by JAK2V617F.

29 In mouse models, MF occurs mainly secondary to essential thrombocythemia (ET)- or 30 polycythemia vera (PV)-like disorders induced by Jak2V617F or CALRdel52 mutation. Direct and 31 strong activation of the thrombopoietin receptor MPL signaling by MPLW515L or high 32 thrombopoietin levels also results in rapid MF development.1-8 All these models together with 33 the topology of the bone marrow (BM) at the onset of MF underscore the key role of the 34 megakaryocytes (MKs) in the disease. Presently, there is no curative therapy in MF, except 35 allogenic BM transplantation; JAK inhibitors being not selective of the oncogenic JAK2 36 signaling.9 Therefore, it is of great interest to identify and inhibit specific downstream targets 37 deregulated by the abnormal JAK2 activation.

38 Targeting CDK6, which is up-regulated in MPN hematopoietic progenitors, either 39 pharmacologically by Palbociclib or by its genetic deletion in Jak2V617F and MPLW515L mice 40 significantly inhibits MPN development.10,11 In addition, the knock-out (KO) of p19 Ink4d, a 41 CDK4/CDK6 inhibitor that regulates the endomitotic arrest of MKs,12 leads to the amplification 42 of hyperploid MKs, increased levels of TGF-β1 and fibrosis development with aging.13

43 Here, we first investigated p19<sup>ink4d</sup> and CDK6 expressions in MKs of induced 44 Jak2<sup>FLEXV617F/+</sup>SCL-Cre<sup>+/-</sup> (KI thereafter) mice<sup>2</sup>. The p19<sup>ink4d</sup> expression level decreased unlike 45 that of CDK6 in MKs from KI mice as compared to controls (<strong>SF 1A</strong>) and correlated with their 46 higher ploidy (major peak at 32N <em>versus</em> 16N) (<strong>SF 1B</strong>). These results suggest that p19<sup>ink4d</sup> 47 downregulation could contribute to the abnormal megakaryopoiesis and MF development in 48 the Jak2<sup>V617F</sup> context.

49
To further support this hypothesis, the *p19<sup> Ink4d-/-</sup>* (*p19* KO)<sup>14</sup> and KI mice were crossed and heterozygous expression of *Jak2<sup>V617F</sup>* was induced in native *p19<sup> Ink4d-/-/Jak2<sup>FLEXV617F/+</sup>SCL-Cre<sup>+/-</sup>* (KOKI thereafter) and KI mice. No marked difference in survival curve and blood parameters was detected between KI and KOKI mice (**SF2A**, **B**). However, as soon as 1-week post-*Jak2<sup>V617F</sup>* induction, there were a significant reduction of BM cellularity and a faster development of splenomegaly in KOKI compared to KI mice (**Fig 1A**, **B**). Moreover, after 3 weeks of *Jak2<sup>V617F</sup>* induction, a grade 1 BM MF was already present in KOKI mice (**Fig 1C**, **SF 2C**). At 8 weeks, we detected a grade 2/3 MF in the BM of all KOKI mice compared to a grade 1 MF in some, but not all KI mice (**Fig 1C**, **SF 2C**). A similar accelerated progression of fibrosis was seen in the spleen (**SF 2D**). Numerous dysplastic MKs in both BM and spleen were visible (**SF 3A**, **B**). *P19<sup> Ink4d</sup>* deletion accelerated the development of *Jak2<sup>V617F</sup>*-dependent extramedullary hematopoiesis (EMH) as illustrated by the increased erythropoiesis and granulopoiesis in the KOKI spleens as early as 1-week post-*Jak2<sup>V617F</sup>* induction (**SF 4A**, **B**, **D**, **E**). At 3 weeks, we observed a marked decrease in all the BM erythroid populations of KOKI mice in accordance with the establishment of MF, compared to KI mice (**SF 4C**).

We next focused on MKs as they represent the major source of TGF-β1 involved in the MF development. The numbers of MK progenitors (MK-Ps) and MKs gradually increased from 1-week post-*Jak2*<sup>V617F</sup> induction (data not shown) to be significant at 3 weeks in the BM and spleen of KI and KOKI mice (**Fig 1D**, **E**, **SF 5A**, **B**) suggesting that *p19<sup>INK4D</sup>* depletion predominantly increases the proliferation of MK-Ps during differentiation. Importantly, a significant increase of MK-Ps and MKs was found in the spleen of KOKI compared to KI mice, consistent with an enhanced EMH (**SF 5C**, **D**). As expected, the study of MK ploidy level showed a major peak at 16N in WT mice and at 64N in *p19* KO mice (**SF 5E*)<sup>12</sup> and the mean ploidy level (&gt;4N) was significantly higher in KOKI than in *p19* KO and KI mice (55.9N, 43.7N and 40.0N, respectively) (**Fig 1F**). These polyploid KOKI MKs were dysplastic and
diffuse von Willebrand factor (vWF) and P-selectin staining (SF 5F) was observed in BM
suggesting a spontaneous activation of MKs with premature release of granule content in KOKI
mice. This was confirmed at 1-week post-Jak2<sup>V617F</sup> induction. Indeed, the KOKI mice showed
the presence of numerous abnormal MKs (Fig 1G) with an increased P-selectin expression at
cell surface (Fig 1H) showing that p19 loss accelerates the development of an abnormal
megakaryopoiesis. Of note, electron microscopy revealed an abnormal localization of granules,
mitochondria and exosome-like particles close to the cell membrane only in KOKI MKs due to
a loss of the marginal zone. In addition, a more important emperipolesis was observed in KOKI
than in KI MKs (SF6A-D). Interestingly, KOKI MKs significantly harbored higher levels of
soluble vWF measured in BM supernatants compared to KI mice, confirming their activation
(Fig 1I). Total and active TGF-β1 concentration measured in BM supernatants were increased
in both genotypes without significant difference between KOKI and KI mice (SF 6E).
Nevertheless, only BM supernatants from KOKI mice co-cultured with stromal MS-5 cells
induced a significant increase in TGF-β1-mediated target genes such as Actin alpha2 (Acta2),
Plasminogen Activator Inhibitor 1 (Pai1) and an increased trend in Connective tissue growth
factor (Ccn2) (SF 6F). These results suggest that active TGF-β1 levels might be underestimated
in KOKI BM potentially due to its rapid receptor binding, signaling and internalization in
stromal cells, leading to a decreased bioavailability.

Overall, these data show that KOKI mice present an accelerated development of MF as
compared to KI mice that were shown to lose approximately half of p19 expression. This
accelerated MF in KOKI as compared to KI mice is associated with numerous dysplastic,
activated, and hyperploid MKs leading to premature release of granule content such as vWF
and TGF-β1.
P19<sup>INK4d</sup> is an inhibitor of CDK4/6, but has also other functions. Thus, we used Palbociclib (PB), a CDK4/6 inhibitor, to investigate if CDK4/6 activation was involved in the MF triggered by Jak2<sup>V617F</sup> and p19<sup>INK4d</sup> loss.

After 2 weeks of treatment, PB induced a decrease in the blood parameters (Fig 2A) of the KOKI mice, and only slightly decreased their already low BM cellularity. In contrast, a 2.1-fold decrease was observed in WT mice due to a reduction of all types of progenitors (Fig 2B, SF 7A-D). Interestingly, PB restored normal spleen weight in KOKI mice (Fig 2C) due to a decrease in the numbers of all progenitors (SF 7D-G). In KOKI BM, PB treatment induced an increase in LSK number and a decrease in MK-Ps with no change in erythroid progenitors (pre-CFU-E) and granulo-monocytic progenitors (GMP) (SF 7A-D). Although MK number in KOKI BM did not change (SF 8A), PB decreased their modal ploidy (64N versus 32N) (SF 8B) and mean ploidy (65.3N versus 35.2N) (Fig 2D). PB-treated KOKI MKs were of smaller size with a less diffuse vWF staining in the cytoplasm as compared to untreated condition (SF 8C). Importantly, PB treatment prevented the development of a reticulin fiber network (Fig 2E) and restored basal active TGF-β1 levels (Fig 2F) in the KOKI BM. Overall, the PB treatment prevented the occurrence of EMH and the development of MF in KOKI mice through inhibition of MK endomitosis and TGF-β1 activation.

To further confirm the effect of PB on KOKI and evaluate its role in KI mice, we used a model of primary transplanted mice, with BM cells of the different genotypes (WT, KI and KOKI) being engrafted into lethally-irradiated WT mice. In both KI and KOKI models, PB administration decreased blood parameters (SF 9A), BM cellularity in all the mice except in KOKI mice (SF 9B), and spleen weight particularly in KI and KOKI mice (SF 9C). A decrease in MK-Ps due to an inhibition of cell cycle and in mean ploidy level of MKs was detected in both KI and KOKI animals while MK numbers were only reduced in KI mice (SF 9D-G). The MKs were smaller as shown by vWF staining, and the MF development in the BM of KOKI
mice was prevented by PB treatment (SF 9H). Overall, these results show a similar effect of PB on hematopoietic parameters in KI and KOKI mice. To further study the specificity of PB treatment on Jak2<sup>V617F</sup> cells, a competitive engraftment between 75% WT-GFP<sup>+</sup> cells and 25% of KI- or KOKI-GFP<sup>+</sup> cells was performed. In both WT/KI and WT/KOKI engrafted mice, PB treatment once again significantly reduced the blood parameters (SF 10A). To know whether this effect was due to the specific targeting of Jak2<sup>V617F</sup> cells, the frequencies of GFP<sup>+</sup> cells were analyzed. The PB treatment significantly decreased the proportion of Jak2<sup>V617F</sup> granulocytes, platelets and erythrocytes in mice engrafted with WT/KI and WT/KOKI cells (Fig 2G, SF 10B). A significant decrease in the BM cellularity (SF 10C) and spleen weight (SF 10D) was associated with a decrease in the frequencies and numbers of Jak2<sup>V617F</sup> MKs, erythroblasts and granulocyte precursors (Fig 2H). A specific decrease in the frequencies and numbers of Jak2<sup>V617F</sup> SLAM and MK-Ps in both WT/KI and WT/KOKI mice was also detected (SF 10E).

Finally, to test whether these last results could be extrapolated to human, we tested the effect of PB treatment on the clonogenicity of JAK2<sup>V617F</sup> progenitors. We observed a decrease in the clonogenicity of the JAK2<sup>V617F</sup> CD34<sup>+</sup> cells isolated from peripheral blood of 3 ET and 2 PV patients (JAK2<sup>V617F</sup> variant allele frequency 4.4-40.5%) (Fig 2I).

Overall, these results afford evidence that p19<sup> Ink4d </sup> deregulation is involved in the abnormal megakaryopoiesis of Jak2<sup> V617F </sup> mice and that its loss accelerates the development of a severe myelofibrosis. The effects of p19<sup> Ink4d </sup> deregulation are mediated through CDK4/6 overactivation. Therefore, CDK4/CDK6 inhibitors appear as potent therapeutic molecules to prevent the progression of MPN into MF. Preclinical studies have shown their synergic effect with ruxolitinib, which predominantly targets the inflammation.<sup> 10,15 </sup>

145
146
147
148
149 Acknowledgments

150 The authors thank P. Rameau, C. Catelain, Y. Lecluse, T. Manoliu from the Imaging and
151 cytometry platform PFIC and S. Souquere from Electronic microscopy platform, UMS3655-
152 AMMICA, Gustave Roussy Villejuif, France; to O. Bawa, H. Rocheteau and N. Signolle for
153 immunohistochemistry and immunofluorescence, and C. Metereau, L. Touchard for help with
154 experiments on animals from preclinical research platform, PETRA, Gustave Roussy Villejuif,
155 France; and to E. Pascal from INSERM U1176 for vWF dosage.

156 This work was supported by grants from Ligue Nationale Contre le Cancer (Equipe labellisée
157 2016, 2019 and 2022 to HR) and MPN research foundation (IP). LG was supported by a
158 fellowship from Association pour la recherche contre le cancer (ARC, France).

159 Authorship Contributions

160 HD, DM, LG, ALC, MEK, AK and IAD performed and analyzed experiments. FP provided
161 patient samples. HD, DM, IAD, LG, ND, AK, FP, CM, RG, MW, VW, IP, HR discussed
162 results. HR supervised the work. HD, DM, LG, VW, IP and HR wrote the article. DM and LG
163 equally contributed to this work.

164 Disclosure of Conflict of Interest: The authors declare that there are no conflicts of interest.

165

166

167

168

169

170
171 References

1. Akada H, Yan D, Zou H, Fiering S, Hutchison RE, Mohi MG. Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease. *Blood* 2010 Apr 29; **115**(17): 3589-3597.

175

2. Hasan S, Lacout C, Marty C, Cuingnet M, Solary E, Vainchenker W, et al. JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNalpha. *Blood* 2013 Aug 22; **122**(8): 1464-1477.

179

3. Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. *PLoS Med* 2006 Jul; **3**(7): e270.

183

4. Lacout C, Pisani DF, Tulliez M, Gachelin FM, Vainchenker W, Villeval JL. JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myelofibrosis. *Blood* 2006 Sep 1; **108**(5): 1652-1660.

187

5. Marty C, Pecquet C, Nivarthi H, El-Khoury M, Chachoua I, Tulliez M, et al. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. *Blood* 2016 Mar 10; **127**(10): 1317-1324.

191

6. Malara A, Gruppi C, Abbonante V, Cattaneo D, De Marco L, Massa M, et al. EDA fibronectin-TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis. *J Exp Med* 2019 Mar 4; **216**(3): 587-604.

195

7. Villeval JL, Cohen-Solal K, Tulliez M, Giraudier S, Guichard J, Burstein SA, et al. High thrombopoietin production by hematopoietic cells induces a fatal myeloproliferative syndrome in mice. *Blood* 1997 Dec 1; **90**(11): 4369-4383.

199

8. Wagner-Ballon O, Chagraoui H, Prina E, Tulliez M, Milon G, Raslova H, et al. Monocyte/macrophage dysfunctions do not impair the promotion of myelofibrosis by high levels of thrombopoietin. *Journal of immunology (Baltimore, Md : 1950)* 2006 Jun 1; **176**(11): 6425-6433.

204

9. Verstovsek S, Kantarjian HM, Estrov Z, Cortes JE, Thomas DA, Kadia T, et al. Long-term outcomes of 107 patients with myelofibrosis receiving JAK1/JAK2 inhibitor ruxolitinib: survival advantage in comparison to matched historical controls. *Blood* 2012 Aug 9; **120**(6): 1202-1209.

209

10. Dutta A, Nath D, Yang Y, Le BT, Mohi G. CDK6 Is a Therapeutic Target in Myelofibrosis. *Cancer research* 2021 Aug 15; **81**(16): 4332-4345.

212

11. Uras IZ, Maurer B, Nivarthi H, Jodl P, Kollmann K, Prchal-Murphy M, et al. CDK6 coordinates JAK2 (V617F) mutant MPN via NF-kappaB and apoptotic networks. *Blood* 2019 Apr 11; **133**(15): 1677-1690.

216

12. Gilles L, Guieze R, Bluteau D, Cordette-Lagarde V, Lacout C, Favier R, et al. P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1. *Blood* 2008 Apr 15; **111**(8): 4081-4091.

220
221 13. Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, et al. p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging. *Stem Cell Reports* 2014 Dec 9; **3**(6): 1085-1102.

224

225 14. Zindy F, van Deursen J, Grosveld G, Sherr CJ, Roussel MF. INK4d-deficient mice are 226 fertile despite testicular atrophy. *Mol Cell Biol* 2000 Jan; **20**(1): 372-378.

227

228 15. Rampal RK, Pinzon-Ortiz M, Somasundara AVH, Durham B, Koche R, Spitzer B, et 229 al. Therapeutic Efficacy of Combined JAK1/2, Pan-PIM, and CDK4/6 Inhibition in 230 Myeloproliferative Neoplasms. *Clin Cancer Res* 2021 Jun 15; **27**(12): 3456-3468.

231
258 magnification. The white arrow indicates the MK with disorganized cytoplasm. H. P-selectin
259 expression on cell surface of mature BM MKs (CD41⁺CD42⁺). The median fluorescence
260 intensity (MFI) for each mouse of each genotype was normalized to the MFI measured for P-
261 selectin on MKs of one WT mouse. I. % of vWF measured by ELISA in BM supernatants of
262 WT, KI and KOKI mice. H-I. Data represent mean±SEM, *p<0.05; **p<0.01, nonparametric
263 Mann-Whitney test. WT (n=6), KI (n=5), KOKI (n=6).

264

265 **Figure 2. Palbociclib treatment prevents myeloproliferation, splenomegaly and MF**
266 **development in Jak2<sup>V617F</sup> context.**

267 **A-F.** Two weeks after Jak2<sup>V617F</sup> induction, KOKI mice were treated daily for 2 weeks with PB
268 150 mg/kg, a dose that did not affect the mouse weight (SF 11). The WT mice were used as
269 controls. **A.** White blood cells count (WBC) (x10<sup>3</sup>/µL), platelet count (x10<sup>3</sup>/µL), Hematocrit
270 (%) and Hemoglobin (g/dL) were analyzed weekly from week 0 to week 4 post-Jak2<sup>V617F</sup>
271 induction (end of treatment and sacrifice). Data represent mean±SEM, *p<0.05; **p<0.01;
272 ***p<0.005, one-way ANOVA coupled to Bonferroni test. WT + vehicle (n=11) or PB (n=11),
273 KOKI + vehicle (n=9) or PB (n=11). **B.** Total BM cellularity for 2 femurs and 2 tibias (x10<sup>6</sup>).

274 **C.** Spleen weight (mg). **B-C.** Data represent mean ± SEM, *p<0.05; **p<0.01; ***p<0.005,
275 nonparametric Mann-Whitney test. WT+vehicle (n=6, 9); WT+PB (n=8,11), KOKI+vehicle
276 (n=6, 9), KOKI+PB (n=7, 11). **D.** Mean ploidy >4N for BM MKs. Data represent median with
277 95%CI, *p<0.05, nonparametric Mann-Whitney test. n=3-4 for each condition. **E.** Left panel:
278 Representative pictures of KOKI BM stained with a standard histological silver kit to
279 underscore reticular fibers. Scale bar = 20 µm. Right panel: Fibrosis grade scored in BM. Data
280 represent mean± SEM, **p<0.01, nonparametric Mann-Whitney test. WT+vehicle (n=3),
281 WT+PB (n=3), KOKI+vehicle (n=6), KOKI+PB (n=3).

282 **F.** Total and active concentration of
283 TGF-β1 in BM supernatant. Total concentration is measured after latent TGF-β1 activation
with HCl. Data represent mean±SEM, *p<0.05; **p<0.01, nonparametric Mann-Whitney test.
WT+vehicle (n=4) or PB (n=5), KOKI+vehicle (n=6) or PB (n=5). G-I. PB specifically targets
the Jak2<sup>V617F</sup> clone. G-H. The irradiated (9.5 Gy) WT mice were engrafted with 3x10<sup>6</sup> cells
(75% WT-GFP+ + 25% KI- or KOKI-GFP). After 4 weeks of hematopoietic reconstitution,
Jak2<sup>V617F</sup> was induced by tamoxifen. From week 2 to 6 post-Jak2<sup>V617F</sup> induction, mice were
treated daily with vehicle or 125 mg/kg PB (dose not toxic for engrafted animals, SF 11) and
analyzed at the end of treatment. G. Frequency of Jak2<sup>V617F</sup> (GFP) granulocyte precursors (Gr-
1<sup>+</sup>CD11b<sup>+</sup>), platelets (CD41<sup>+</sup>), and erythrocytes (Ter-119<sup>+</sup>) were analyzed weekly from week
0 to week 6 post-Jak2<sup>V617F</sup> induction. Data represent mean±SEM, **p<0.01; ***p<0.005, one-
way ANOVA coupled to Bonferroni test. KOKI+vehicle (n=8), KOKI+PB (n=9), KI+vehicle
(n=9), KI+PB (n=9). H. Frequencies and absolute numbers of BM Jak2<sup>V617F</sup> MKs
(CD41<sup>+</sup>CD42<sup>+</sup>), erythroblasts (CD71<sup>+</sup>Ter-119<sup>+</sup>) and granulocyte precursors (Gr-1<sup>+</sup>CD11b<sup>+</sup>).
Absolute numbers are calculated according to the total frequency of the population of interest
and the total BM cellularity. Data represent mean±SEM, ns: non-significant *p<0.05;
**p<0.01; ***p<0.005, nonparametric Mann-Whitney test. KOKI+vehicle (n=8), KOKI+PB
(n=9), KI+vehicle (n=9), KI+PB (n=9). I. CD34<sup>+</sup> cells isolated from ET and PV patients were
cultured at 1 progenitor/well in 96-well plates in presence of EPO, TPO, FLT3-L, IL-3, G-CSF,
IL-6, SCF and GM-CSF without or with PB (0.1 µM) for 14 days. Then the individual colonies
were genotyped for JAK2<sup>V617F</sup> or wild-type (WT) JAK2 status for 3 ET and 2 PV patients with
heterozygous JAK2<sup>V617F</sup> mutation with a VAF 4-40.5%. Shown is a ratio of the colony numbers
grown in presence of PB normalized to the colony numbers grown in absence of PB (vehicle).
Data represent mean±SEM, ns: non-significant; **p<0.01, nonparametric Mann-Whitney test.
<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>
311 Supplementary Informations

312
313 MATERIAL AND METHODS

314
315

Patients
---

316 Peripheral blood samples were collected from patients with their written informed consent in
317 accordance with the Declaration of Helsinki and the study was approved by the Ethics
318 Committee from Gustave Roussy; from Comité de Protection des Personnes (CPP) Ile de
319 France IV- Institutional review board (agreement from US Department of Health and Human
320 Services n°IRCB 00003835- Protocol 2015/59-NICB and then n°IRCB 2021-A03067-34.) The
321 JAK2<sup>V617F</sup> VAF (4.4-40.5%) was determined on granulocytes by PCR (patient N#1) or by NGS
322 (patients N#2-5). The sampling was done during the treatment with hydroxyurea or aspirin and
323 the additional mutations are listed in ST1.
324

324
325
**Patient cell purification**
326

326 Mononuclear cells were purified from peripheral blood cells by a Ficoll density gradient, and
327 CD34+ cells were purified by a double-positive magnetic cell sorting system (AutoMACS;
328 Miltenyi Biotec).

329 Mice

330 Heterozygous p19<sup>Ink4d+/-</sup> mice with a C57BL/6 x Sv129j genetic background were kindly
331 provided by Martine Roussel (Memphis, TN) and crossed to obtain homozygous p19<sup>Ink4d+/-</sup> and
332 p19<sup>Ink4d-/-</sup> mice. Homozygous p19<sup>Ink4d-/-</sup> mice were crossed with the conditional
333 Jak2<sup>FLEXV617F/+</sup> SCL-Cre<sup>+/-</sup> KI mice available in the laboratory. Transgenic mice expressing GFP
334 (UBI-GFP/BL6)1 were used in competitive assays.

335 All animal experiments were performed in accordance with 2010/63/UE European legislation
336 and decree n°2013-118 of French legislation; and recorded under protocol number 2012_060
337 and 2020_007_23589. Mice were housed in pre-clinical platform of Gustave Roussy Animal
338 Facilities PFEP (Villejuif, France) (ministerial approval n° 94-076-11) under 12-hour light/12-
339 hour dark cycles and monitored ambient temperature (21 ± 1 °C) and humidity range (20-70%).
340

340
341
**Mice genotyping**

342 Mice were genotyped using the Mouse Direct PCR Kit according to manufacturer's protocol
343 (Bimake) with the different sets of primers available upon request.

344 Jak2<sup>V617F</sup> induction by tamoxifen

345 Tamoxifen (Sigma) solution was administrated to 6-8- weeks-old mice at 200 mg/kg by force-
346 feeding during 4 days. Mice were subsequently fed during 2-4 weeks with tamoxifen food
347 (Ssniff Diet + 400 mg/kg tamoxifen (Sigma)).

348 Blood parameters and mouse experimentation

349 Blood parameters were determined using an automated blood counter (MS9, Schloessing
350 Melet). BM cells were collected by flushing the femurs and two tibiae of individual mice.
351 Cellularity was calculated using the 2 femurs and 2 tibias or 1 femur of mice as indicated in
352 legends. Spleens were weighted and dissociated to prepare single-cell suspensions. The BM
353 and spleen cell number was determined by Sysmex Cell Counter.
354

354
355 **Cell analysis by flow cytometry**

356 BM lineage-negative (Lin⁻) cells were identified for their absence of labeling with the following
357 FITC-conjugated antibodies (Ab) against CD3, Ter-119, CD11b, Gr-1, B220 (BioLegends).
358 APC-conjugated antibodies for GFP positive mice were used in competitive engraftment
359 experiments. BM and spleen cell content were investigated including: progenitor cell-enriched
360 fraction Lin⁻Sca-1⁺c-Kit⁺ (LSK), hematopoietic stem cell (HSC)-enriched population Lin⁻Sca-
361 1⁺c-Kit⁺CD48⁻CD150⁺ (SLAM), and progenitors from the megakaryocytic (MK-P: Lin⁻Sca-1⁻
362 c-Kit⁺CD150⁺CD41⁺), erythroid (pre-CFU-E: Lin⁻Sca-1⁻c-Kit⁺CD41⁻CD150⁺FcγRII/III⁻
363 CD105⁺) and granulocytic (GMP: Lin⁻Sca-1⁻c-Kit⁺CD41⁻FcγRII/III⁺CD150⁻) lineages, using
364 the anti-c-Kit-PerCP/Cy5.5, Sca-1-PE/Cy7, -CD150-PE, -CD41-BV421, -CD105-APC, -
365 CD16/32-APC/Cy7 and -CD48-Pacific Blue Ab (all purchased from BioLegend).

366 Precursor and mature cells including MKs (CD41⁺CD42⁺), granulocytes (Gr1⁺CD11b⁺) and the
367 erythroid lineage (CD71⁺Ter119⁻, CD71⁺Ter119⁺, CD71⁻Ter119⁺), were analyzed with Ab
368 against CD41-PerCP/Cy5.5, CD42d-APC, Gr1-FITC, Cd11b-PE, Ter119-FITC (BioLegend),
369 CD71-PE (BD).

370 Activation of MKs, was investigated by determining the level of P selectin on the cell surface.
371 In this purpose, the Median Fluorescent Intensity (MFI) of anti-P-selectin-PE Ab (Emfret) and
372 of IgG-PE at cell surface (without activation) was measured on cells expressing CD41 and
373 CD42 (anti CD41-PerCP/Cy5.5 and CD42d-APC antibodies) after Lin positive depletion.
374 Treatment of cells with TRAP 4 at 10mM was used as a positive control of activation.

375 **Progenitors cell cycle analysis by flow cytometry**

376 BM progenitors were first labelled with non-conjugated antibodies against CD3-FITC, Ter-
377 119-FITC, Cd11b-FITC, Gr-1-FITC, B220-FITC, then with c-kit PerCP/Cy5.5, Sca-1-PE/Cy7,
378 CD41-AF700R, CD48-APC/Cy7 and CD150-PE (BioLegend). After extracellular staining,
379 cells were fixed and permeabilized with Cytofix/Cytoperm and cytofix plus (BD Biosciences).
380 The intracellular staining was then performed with a Ki-67-APC Ab (Biolegend) and with
381 DAPI (5 µg/mL). Different cell cycle phases were analyzed by flow cytometry.

382 **Flow Cytometry analysis and cell sorting**

383 FACSCanto II, FACS Canto X and LSR Fortessa (BD Biosciences) were used for analysis. All
384 data were collected on Diva software. INFLUX (BD Biosciences) was used for cell sorting.

385 **MK ploidy analysis**

386 BM MKs were labeled with anti-CD41-FITC and anti-CD42d-APC Ab (BioLegend). After
387 hypotonic lysis with a citrate solution containing 50 µg/mL propidium iodide (PI) (Sigma-
388 Aldrich), ploidy was determined using a FACSCanto II cytometer (BD Bioscience). The mean
389 ploidy in >4N MKs was calculated as (%8Nx0.08) + (%16Nx0.16) + (%32Nx0.32) +
390 (%64Nx0.64) + (%128Nx1.28) + (%256Nx2.56).

391 **Histology and immunohistochemistry**

392 BM (femur) and spleen samples were fixed in 4% paraformaldehyde (PFA), decalcified and
393 embedded in paraffin. Sections were stained with hematoxylin, eosin and safran. MK were
394 revealed with immunohistochemistry using a rabbit anti-von Willebrand Factor (vWF) antibody
395 (Dako). Reticulin fibers were stained following the Gordon and Sweet's silver staining
396
397

procedure (black stain). Slides were examined using a Zeiss Axiophot microscope (Carl Zeiss) with 2.5×1 (magnification ×2.5) or 10×/1 (magnification ×10) numeric aperture objectives.

398

## Immunofluorescence

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401
402
403
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407

BM (femur) and spleen samples were fixed in 4% paraformaldehyde (PFA), decalcified and embedded in paraffin. After deparaffinization and rehydration, the antigen retrieval was performed (Tris EDTA pH8, 98°C, 30min). Slides were cooled 30 min at room temperature and after washing in distilled water they were permeabilized (4% Triton X-100 in PBS (PBS-T)) and blocking of non-specific binding was done with 5% BSA in PBS-T for 10 min. Then the slides were incubated with specific anti-vWF-FITC (Abcam) and P-selectin-PE (Emfret) antibodies diluted in blocking solution (1% BSA in PBS-T) for 1h at room temperature. Nuclear labelling was done with DAPI (Molecular Probes). The slides were examined using Vectra Polaris Automated Quantitative Pathology Imaging System (AKOYA) with a x20 objective.

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414
415

## Transmission electron microscopy

408
409
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Tibias were flushed with 2% glutaraldehyde in 0.1M phosphate buffer for 1h at 4°C for fixation and were postfixed with 2% osmium tetroxide in 0.1M phosphate buffer for 1h at room temperature. Following dehydration through a graded ethanol series, samples were embedded in Epon™ 812. Polymerization was complete after 48 h at 60 °C. Ultrathin sections were stained with standard uranyl acetate and lead citrate and observed with FEI Tecnai 12 electron microscope. Digital images were taken with a SIS MegaviewIII CCD camera.

416

## In vitro growth of MKs in semi-solid conditions

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418
419
420

To quantify MK progenitors (CFU-MK), 1x10⁵ total BM cells were seeded in serum-free fibrin clot media as previously described² supplemented with TPO (10 ng/mL, Kirin Brewery, Tokyo, Japon), mSCF (25ng/mL, Biovitrum AB, Stockholm, Sweden) and IL-6 (10ng/mL, Peprotech, France).

421
422
423

After 7 days of culture, cells were fixed with glutaraldehyde 5% and MK colonies were stained with acetylcholinesterase solution (acetylcholine iodide dissolved in sodium phosphate buffer pH 6.3, 0.1 M sodium citrate, 30 mM copper sulfate, 5mM potassium ferricyanide).

424
425
426

## TGF-β1 and vWF level determination in BM supernatant

BM cells were recovered by flushing one femur in 1 mL of PBS and centrifuged.

426

### TGF-β1

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428
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430
431

a) TGF-β1 level in the supernatant was detected using the Quantikine ELISA Kit (R&D system, Bio-Techne SAS). The kit detects active TGF-β1 levels. In order to measure latent TGF-β1 levels, we followed the manufacturer's instruction to activate the latent form by addition of 20% of 1N HCl followed with neutralization with 15% of 1.2N NaOH/0.5 M HEPES. Total TGF-β1 represents the addition of latent and active forms.

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b) 40 x 10³ of BM stromal murine MS-5 cells were co-cultured in 6-well plates with 200µl of BM supernatants and 800 µl of DMEM supplemented with 10% FCS at 37°C in 5% CO₂ for 5 hours. The expression levels TGF-β1 responding genes (Ccn2, Acta1 and Pai1) were then measured by qPCR.³
436 vWF
437 vWF antigen levels in BM supernatants were measured with an in-house enzyme-linked
438 immunosorbent assay essentially as previously described⁴. A pooled plasma prepared from 20
439 (males and females) C57Bl/6 mice was used as reference.
440

440

441 Transplant procedures

442 Recipient are WT mice irradiated at 9.5Gy with XRAD 320 KV irradiator. These mice were
443 grafted with 3x10⁶ (15x10⁶ cells/mL in PBS) WT or non-induced KOKI BM cells by retro
444 orbital injection. For competitive procedure mice have received 75% of WT-GFP cells + 25%
445 of non-induced KI Jak2<sup>V617F</sup> or 25% of non-induced KOKI cells. Proportions of cells have been
446 verified by following the GFP by flow cytometry. Four weeks after engraftment, Jak2<sup>V617F</sup>
447 expression was induced by tamoxifen.

448 Palbociclib administration

449 Palbociclib isethionate salt (#P-7766, LC Laboratories, USA) was suspended in sterile water (15 or 12.5 mg/mL) and 200 µL/20g were administered to the mice every 24 h by force-feeding.
450 Preventive treatment was administrated between weeks 2 and 4 post- Jak2<sup>V617F</sup> induction at 451 150mg/kg for native mice. For engrafted mice, PB was administrated between weeks 2 and 6 452 post-Jak2<sup>V617F</sup> induction at 125 mg/kg (or at the indicated concentration in the legend). Control 453 mice received vehicle (water). The toxicity of Palbociclib was tested in both native and 454 engrafted mice (SF11). 455

455

456 Patient progenitor cultures

457 To study the effect of Palbociclib, CD34⁺ cells were sorted (anti-CD34-APC Ab (BD) and DAPI) at 1 progenitor/well in 96-well plates with a BD Influx cell sorter. Clones were 458 expanded in serum-free medium with a cocktail of human recombinant cytokines containing 1 459 U/mL EPO (Amgen), 20 ng/mL TPO (generous gift from Kirin), 10 ng/mL FLT3-L (Celldex 460 Therapeutics), 10 ng/mL IL-3, 20 ng/mL G-CSF, 10 ng/mL IL-6 (Miltenyi Biotech), 25 ng/mL 461 SCF and 5µg/mL GM-CSF (Peprotech) without or with Palbociclib (0.1 µM). Fourteen days 462 later, individual colonies corresponding to the progeny of each progenitor were lysed and DNA 463 was genotyped for homozygous, heterozygous or wild-type (wt) JAK2<sup>V617F</sup> status as previously 464 described⁵. 465

460

461

462

463

464

465

466 qPCR

467 Sorted BM MKs and MS-5 cells cocultured with BM supernatants were kept in Trizol and RNA
468 was extracted with Direct zol RNA Microprep. *p19<sup>lnk4d</sup*, *CDK6*, *Ccn2*, *Acta2* and *Pai1*
469 expression levels were measured by RT-qPCR (Superscript IV Vilo and Takara Sybr Green).
470 HPRT was used as housekeeping gene. Following murine primers were used: *p19<sup>lnk4d</sup>*: F: CAA-
471 GAT-GCC-TCC-GGT-ACT-AG and R: CAA-CTC-CAG-GGG-AGT-GAG-AC; *Cdk6*: F:
472 GGCACCTGGAGACCTTCGA and R: CGTGACACTGTGCACACATCA; *Ccn2*: F: AGG
473 ACC GCA CAG CAG TTG and R: GCA GTT GGC TCG CAT CAT AG; *Acta2*: F: TCA GGG
474 AGT AAT GGT TGG AAT GG and R: GTT GGT GAT GAT GCC GTG TTC; *Pai1*: F: CTC
475 CTC ATC CTG CCT AAG TTC and R: GTC CCG CTC TCG TTT ACC.

468

469

470

471

472

473

474

475

476 Cytometry data and statistical analyses.

477 Cytometry data were analyzed on FlowJo software and statistical analyses were performed
478 using the Prism software. If not otherwise mentioned, tests used an unpaired design with two-
479 tailed nonparametric Mann-Whitney test. Blood parameters were analyzed with the One-way
ANOVA with the Bonferroni correction test. Results are presented as means ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001; ****p<0,0001; “n =” stands for individual mice in all experiments.

**Data sharing statement**

Other material and methods will be provided upon request by corresponding author.

484

485

486 Supplementary references

487

1. Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. *Cell Immunol.* **214**:110-22 (2001).

490

2. Debili N, Coulombel L, Croisille L, Katz A, Guichard J, Breton-Gorius J, et al. Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow. *Blood* **88**:1284-1296 (1996).

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3. Lambert J, Saliba J, Calderon C, Sii-Felice K, Salma M, Edomond V, et al. PPARγ agonists promote the resolution of myelofibrosis in preclinical models. *J Clin Invest.* 131(11):e136713 (2021).

497

4. Lenting PJ, Westein E, Terraube V, Ribba AS, Huizinga EG, Meyer D, et al. An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation. *J Biol Chem.* 279:12102-9. (2004).

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5. Delhommeau F, Dupont S, Tonetti C, Massé A, Godin I, Le Couedic JP, et al. Evidence that the JAK2 G1849T (V617F) mutation occurs in a lymphomyeloid progenitor in polycythemia vera and idiopathic myelofibrosis. *Blood* **109**, 71-77 (2007).

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508
Supplementary figures

Supplementary figure 1

A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

SF1: Ploidy and *p19<sup>lnk4d</sup>* and *CDK6* expression levels in WT and *Jak2<sup>V617F</sup>* (KI) MKs.

A. *p19<sup>lnk4d</sup>* and *Cdk6* expression relative to HPRT in WT and KI CD41<sup>+</sup>CD42<sup>+</sup> MKs after 3 weeks post-*Jak2<sup>V617F</sup>* induction. Data represent mean±SEM, \*p<0.05; \*\*p<0.01; \*\*\*p<0.005, nonparametric Mann-Whitney test. WT (n=4, 5) and KI (n=5). B. The representative picture of BM MK ploidy for WT and KI mice.
<figure><img src="image_6.png" /><figcaption>Supplementary figure 2</figcaption></figure>

SF2. P19<sup>Ink4d</sup> depletion favors the rapid development of splenomegaly and myelofibrosis in Jak2<sup>V617F</sup> context

A. Survival curve (315 days follow-up) for the WT, *p19* KO (KO p19), *Jak2*<sup>V617F</sup> KI (KI) or *p19* KO/*Jak2*<sup>V617F</sup> KI (KOKI) mice according to the days post-*Jak2*<sup>V617F</sup> induction. n=7-11 for each genotype. Log-rank test was used, \*p<0.05; \*\*p<0.005; \*\*\*p<0.001. B. Platelet count (x10<sup>3</sup>/µL), white blood cells count (WBC, x10<sup>3</sup>/µL), hematocrit (%), hemoglobin (g/dL) and mean corpuscular (µm<sup>3</sup>) were analyzed weekly for 11 weeks post-*Jak2*<sup>V617F</sup> induction. Data represent mean±SEM, \*p<0.05; \*\*p<0.01; \*\*\*p<0.005, one-way ANOVA coupled to Bonferroni test, (n=4 to 29). C. Fibrosis grade scored in bone marrow (BM) of WT (n=3 or 4),
534 KO p19 (n=3), KI (n=3 or 4) and KOKI (n=3 or 4) mice at different weeks (W) post-Jak2<sup>V617F</sup>

535 induction. Data represent mean±SEM, *p<0.05, nonparametric Mann-Whitney test. D.

536 Representative pictures of spleen stained with a standard histological silver kit to reveal

537 reticular fibers at 3 and 8 weeks post-Jak2<sup>V617F</sup> induction. Scale bar = 20µm.

538
<figure><img src="image_7.png" /><figcaption>539 **Supplementary figure 3**</figcaption></figure>

<figure><img src="image_8.png" /></figure>

SF3. A-B. Representative pictures of BM (A) and spleen (B) von Willebrand factor staining at 3 and 8 weeks post-Jak2<sup>V617F</sup> induction. Scale bar = 20µm.

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<figure><img src="image_9.png" /><figcaption>Supplementary figure 4</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

SF4.

P19<sup>Ink4d</sup> depletion favors the rapid development of extramedullar hematopoiesis in Jak2<sup>V617F</sup> context

Erythroid differentiation followed in bone marrow (A, C) and spleen (B, D) with CD71 and Ter119 markers at week 1 (W1; A, B) and 3 (W3; C, D) post-Jak2<sup>V617F</sup> induction. CD71<sup>+</sup>Ter119<sup>−</sup> cells represent the more immature erythroblasts and the CD71<sup>−</sup>Ter119<sup>+</sup> population represents the more mature erythroid cells. E. Absolute numbers of granulocytes (Gr1<sup>+</sup> CD11b<sup>+</sup>) in spleen at week 1 (W1) and 3 (W3) post-Jak2<sup>V617F</sup> induction. A-E. Absolute numbers are calculated according to the total frequency of each population and the total BM or spleen cellularity. Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. WT (n= 6-12), KO p19 (n=7-11), KI (n= 9-11) and KOKI (n=11-16).
A

<figure><img src="image_14.png" /></figure>

B

<figure><img src="image_15.png" /></figure>

C

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /></figure>

E

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

F

<figure><img src="image_20.png" /></figure>

SF5. *P19<sup>Ink4d</sup>* depletion favors the amplification of MK lineage in *Jak2<sup>V617F</sup>* context

**A.** Frequency of MK-Ps (Lin⁻ Sca-1⁻c-kit⁺CD41⁺CD150⁺) in BM 3 weeks post-*Jak2*<sup>V617F</sup> induction. WT (n=10), KO p19 (n=10), *Jak2*<sup>V617F</sup> KI (KI, n=12), *p19* KO / *Jak2*<sup>V617F</sup> KI (KOKI, n=12). **B, D.** The numbers of MK stained by immunohistochemistry with vWF in BM (**B**) and spleen (**D**). The MK numbers were counted on equal area of 3 independent BM and 5 independent spleen pictures for each mouse. WT (n=5), KO p19 (n=4), *Jak2*<sup>V617F</sup> KI (KI, n=4), *p19* KO / *Jak2*<sup>V617F</sup> KI (KOKI, n=5). Data represent mean±SEM, \*p\<0.05; \*\*p\<0.01, nonparametric Mann-Whitney test. **C.** Absolute numbers of MK-Ps in spleen at week 3 post-*Jak2*<sup>V617F</sup> induction. Data represent mean±SEM, \*p\<0.05; \*\*p\<0.01; \*\*\*p\<0.0001, nonparametric Mann-Whitney test. WT (n=8), *p19* KO (n=8), KI (n=8) and KOKI (n=12). **E.** Ploidy of BM MKs is calculated for each genotype after 3 weeks of *Jak2*<sup>V617F</sup> induction. The
571 representative picture for KO p19 and KOKI genotype is shown on the left panel. The

572 representative picture for WT and KI genotype is shown on SF 1B. The mean percentage for

573 each peak (from 2N to 256N) and for each genotype is shown on the right panel. Data represent

574 mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. WT (n=8),

575 *p19* KO (n=10), *Jak2*<sup>V617F</sup> KI (n=12), *p19* KO / *Jak2*<sup>V617F</sup> KI (KOKI, n=9). **F.**

576 Immunofluorescence staining of MKs in BM of WT or KOKI mice 3 weeks post-*Jak2*<sup>V617F</sup>

577 induction. vWF is in red, P-selectin in green and nucleus (DAPI) in blue color. A diffuse

578 localization of vWF and P-selectin are detected in BM of KOKI mice. Scale bar = 20µm.

579
<figure><img src="image_21.png" /><figcaption>580 Supplementary figure 6</figcaption></figure>

<figure><img src="image_22.png" /></figure>
D

<figure><img src="image_23.png" /></figure>

KOKI

<figure><img src="image_24.png" /></figure>

KOKI

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

KOKI

<figure><img src="image_28.png" /></figure>

KOKI

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>
E

<figure><img src="image_31.png" /></figure>

F

<figure><img src="image_32.png" /></figure>

SF6. Abnormal ultrastructure of MKs in KOKI mice with increased TGFb1 secretion.

A-D. Representative pictures of MKs in BM of WT, KI or KOKI mice at 1-week post-Jak2<sup>V617F</sup> induction observed by electronic microscopy. WT (A) and KI (B) MKs with conserved marginal zone are shown. C. Emperipolesis in KI and KOKI MKs indicated by black arrows.

D. KOKI MKs with disrupted marginal zone are shown. Red arrows indicate the granules, yellow arrows the mitochondria and green arrows the exosome-like particles localization on the periphery of cells. The lower panels show zooming in on the black squares of the panels at the top.

The interrupted black lines show the edge of the cell wall. Scale bar = 2, 5 or 10µm as indicated.

E. Total and active concentration of TGF-β1 in BM supernatant after 1 (W1) week post-Jak2<sup>V617F</sup> induction. Total concentration is measured after latent TGF-β1 activation with HCl.

Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. WT (n=6), KI (n=8, 4), KOKI (n=10, 5). F. Ccn2; Acta2 and Pai1 expression relative to Hprt in MS-5 stromal cells co-cultured for 5h with BM supernatants isolated from BM of WT, KI and KOKI mice at 1-week post-Jak2<sup>V617F</sup> induction. Data represent mean±SEM, *p<0.05; nonparametric Mann-Whitney test. WT (n=4), Jak2<sup>V617F</sup> KI (n=4) and KOKI (n=4).

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A

BM

<figure><img src="image_33.png" /></figure>

B

BM

<figure><img src="image_34.png" /></figure>

C

BM

<figure><img src="image_35.png" /></figure>

D

BM

<figure><img src="image_36.png" /></figure>

E

Spleen

<figure><img src="image_37.png" /></figure>

F

Spleen

<figure><img src="image_38.png" /></figure>

G

Spleen

<figure><img src="image_39.png" /></figure>

H

Spleen

<figure><img src="image_40.png" /></figure>

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618

SF7. Palbociclib treatment prevents establishment of extramedullary hematopoiesis 2 weeks after Jak2V617F induction, mice were treated daily with PB (150 mg/kg) for 2 weeks. A-D. Absolute numbers of bone marrow progenitors at the end of treatment with vehicle or PB. A. LSK (Lin⁻Sca-1⁺c-Kit⁺). B. Pre-CFUE (Lin⁻c-kit⁺ Sca-1⁻CD41⁻ FCγR⁻ CD105⁺ CD150⁺). C. GMP (Lin⁻c-kit⁺ CD41⁻ FCγR⁺ CD150⁻). D. MK-P (Lin⁻ Sca-1⁻c-Kit⁺CD150⁺CD41⁺). Data represent mean±SEM, *p<0.05, nonparametric Mann-Whitney test. WT+vehicle (n=3), WT+PB (n=4), KOKI+vehicle (n=3), KOKI+PB (n=4). E-H. Absolute numbers of progenitors in the spleen at the end of treatment. E. LSK. F. Pre-CFU-E. G. GMP. H. MK-P. Data represent mean±SEM, *p<0.05, nonparametric Mann-Whitney test. WT+vehicle (n=3), WT+PB (n=4), KOKI+vehicle (n=3), KOKI+PB (n=4). Absolute numbers were calculated according to the total BM or spleen cellularity.
<figure><img src="image_41.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_43.png" /></figure>

SF8. Palbociclib treatment partially restores megakaryopoiesis in KOKI mice

2 weeks after Jak2<sup>V617F</sup> induction, mice were treated daily with PB (150 mg/kg) for 2 weeks.

A. Absolute numbers of mature MKs (CD41<sup>+</sup>CD42<sup>+</sup>) in BM (1 femur). Data represent mean±SEM, *p<0.05, nonparametric Mann-Whitney test. WT+vehicle (n=3), WT+PB (n=3), KOKI+vehicle (n=4), KOKI+PB (n=4). B. Ploidy analysis: representative histograms of BM MKs for WT or KOKI mice with or without PB treatment. C. Representative pictures of BM von Willebrand factor (vWF) staining for WT or KOKI mice with or without treatment.

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628
629
<figure><img src="image_44.png" /><figcaption>630 Supplementary figure 9</figcaption></figure>

631
632 SF9. Palbociclib treatment prevents the myeloproliferation and restores the
633 megakaryopoiesis in Jak2<sup>V617F</sup> context

634 The irradiated (9.5 Gy) WT mice were engrafted with 3 x10⁶ of total BM WT, Jak2<sup>V617F</sup> KI
635 (KI) or p19 KO / Jak2<sup>V617F</sup> KI (KOKI) cells. After 4 weeks of hematopoietic reconstitution,
636 Jak2<sup>V617F</sup> was induced by tamoxifen. From week 2 to 6 post-Jak2<sup>V617F</sup> induction, mice were
637 treated daily during 4 weeks with vehicle or PB (125 mg/kg) and analyzed at the end of
638 treatment. A. Shown are: white blood cells count (WBC) (x10<sup>3</sup>/µL), platelet count (x10<sup>3</sup>/µL),
639 hematocrit (%), hemoglobin (g/dL) and red blood cells (RBC) count (x10<sup>6</sup>/µL). Data
640 represent mean ± SEM, *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001 one-way ANOVA
641 coupled to
Boneferroni test. WT + vehicle (n=9), WT + PB (n=9), KI + vehicle (n=11), KI + PB (n=10), KOKI + vehicle (n=11), KOKI + PB (n=12). B. Total BM cellularity for 2 femurs and 2 tibiae (x10⁶). C. Spleen weight (mg). B-C. Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. WT+vehicle (n=9), WT+PB (n=9), KI+vehicle (n=11) KI+PB (n=10), KOKI+vehicle (n=11), KOKI+PB (n=12). D-E. Absolute numbers of MK progenitors (MK-P, Lin⁻c-Kit⁺Sca-1⁻CD150⁺CD41⁺) (D), and mature MKs (CD41⁺CD42⁺) (E) in total BM calculated according to their frequency and the total BM cellularity. Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. WT+vehicle (n=8), WT+PB (n=9), KI+vehicle (n=10,11) KI+PB (n=9), KOKI+vehicle (n=10,8), KOKI+PB (n=11,9). F. Mean ploidy level of mature BM CD41⁺CD42⁺ MKs stained with propidium iodide. Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001, t-test Mann-Whitney. WT + vehicle (n=9), WT+PB (n=9), KI+vehicle (n=12), KI+PB (n=10), KOKI+vehicle (n=10), KOKI+PB (n=11). G. Cell cycle analysis of MK-Ps. Gating strategy is shown on the top panel. Histograms on bottom panel represent different cell cycle phases (G0, G1, S/G2/M) in MK-Ps of all genotypes treated with vehicle or PB (125mg/kg). Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric Mann-Whitney test. Statistical analysis represents the significative difference between each cell cycle phase compare to their respective control: PB condition is compared to vehicle condition for same genotype and for same cell cycle phase. For vehicle, different genotypes are compared to the WT for the same cell cycle phase. WT+vehicle (n=8), WT+PB (n=9), KI+vehicle (n=11), KI+PB (n=9), KOKI+vehicle (n=11), KOKI+PB (n=9). H. Left panel: Representative pictures of BM stained with a standard histological silver kit to underscore reticular fibers or von Willebrand factor. Scale bar = 50µm. Right panel: Fibrosis grade scored in bone marrow (BM). Data represent mean±SEM, *p<0.05, nonparametric Mann-Whitney test. KOKI+vehicle (n=3), KOKI+PB (n=5).

667
<figure><img src="image_45.png" /><figcaption>668 Supplementary figure 10</figcaption></figure>

669
670

671 SF10. Palbociclib specifically targets the *Jak2*<sup>V617F</sup> clone

672 Irradiated (9.5Gy) WT mice were engrafted with 3x10<sup>6</sup> cells (75% WT-GFP<sup>+</sup> + 25% KI- or KOKI-GFP<sup>-</sup>). After 4 weeks of hematopoietic reconstitution, Jak2<sup>V617F</sup> was induced by tamoxifen. From week 2 to 6 post-Jak2<sup>V617F</sup> induction, mice were treated daily with vehicle or PB (125mg/kg) and analyzed at the end of treatment. A. Shown are: white blood cells count (WBC) (x10<sup>3</sup>/µL), platelet count (x10<sup>3</sup>/µL), hematocrit (%), hemoglobin (g/dL) and red blood cells count (RBC) (x10<sup>6</sup>/µL). Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005, one-way ANOVA coupled to Bonferroni test. KOKI+vehicle (n=8), KOKI+PB (n=9),
679 KI+vehicle (n=9), KI+PB (n=9). **B.** Gating strategy for the analysis of WT granulocytes

680 (Gr1⁺CD11b⁺GFP⁺), WT erythrocytes (Ter119⁺GFP⁺) and WT platelets (CD41⁺GFP⁺) in

681 peripheral blood. **C.** Total BM cellularity for 2 femurs and 2 tibiae (x10⁶). **D.** Spleen weight

682 (mg). **B, D.** Data represent mean ± SEM, *p<0.05; **p<0.01; ***p<0.005, nonparametric

683 Mann-Whitney test. KOKI+vehicle (n=8), KOKI+PB (n=9), KI+vehicle (n=9), KI+PB (n=8).

684 **E.** Frequencies and absolute numbers of BM Jak2<sup>V617F</sup> progenitors: LSK (Lin<sup>-</sup>Sca-1<sup>+</sup>c-Kit<sup>+</sup>),

685 SLAM (Lin<sup>-</sup>Sca-1<sup>+</sup>c-Kit<sup>+</sup>CD48<sup>-</sup>CD150<sup>+</sup>) and MK-P (Lin<sup>-</sup>Sca-1<sup>-</sup>c-Kit<sup>+</sup>CD150<sup>+</sup>CD41<sup>+</sup>).

686 Absolute numbers are calculated according to the total frequency of the population of interest

687 and the total BM cellularity. Data represent mean±SEM, *p<0.05; **p<0.01; ***p<0.005,

688 nonparametric Mann-Whitney test. KOKI+vehicle (n=8), KOKI+PB (n=9), KI+vehicle (n=9)

689 KI+PB (n=9).
<figure><img src="image_46.png" /><figcaption>691
692 SF11. Toxicity test of Palbociclib treatment</figcaption></figure>

693 A. 2 weeks after Jak2<sup>V617F</sup> induction, WT and p19 KO / Jak2<sup>V617F</sup> KI (KOKI) mice were treated
694 daily with PB (150 mg/kg) for 2 weeks. Mouse weight was followed each week, the histogram
695 represents the percentage of weight loss at the end of 2 weeks of treatment compare to the initial
696 weight. Data represent mean±SEM. WT+vehicle (n=14), WT+PB (n=16), KOKI+vehicle
697 (n=16), KOKI+PB (n=14).
698 Irradiated (9.5Gy) WT mice were engrafted with 3 x10<sup>6</sup> WT
cells. After 4 weeks of hematopoietic reconstitution, mice are treated daily during 6 weeks with
699 vehicle or PB (80 or 150mg/kg). Mouse weight was followed each week, the histogram on the
700 left panel represents the percentage of weight loss at the end of each week of treatment compare
701 to the initial weight. A dose of 150 mg/kg induced a 20% loss of weight and was considered as
702 a toxic in transplanted mice. Spleen and liver weights are shown on the right panel. Data
703 represent mean±SEM, *p<0.05; nonparametric Mann-Whitney test. WT+vehicle (n=4),
704 WT+80 mg/kg PB (n=5), WT+150 mg/kg PB (n=5).
705 The irradiated (9.5Gy) WT mice were
706 engrafted with 3 x10<sup>6</sup> of non-induced KOKI cells. After 4 weeks of hematopoietic
707 reconstitution, Jak2<sup>V617F</sup> was induced by tamoxifen. Mice were treated daily from week 2 to
708 week 6 post-Jak2<sup>V617F</sup> induction with vehicle or PB (80, 100 or 125 mg/kg). Mice weight was
709 followed each week, the histogram on the left panel represents the percentage of weight loss at
710 the end of each week of treatment compare to the initial weight. BM cellularity, spleen and liver
711 weights are shown on the right panel. A decrease in the spleen weight with PB treatment
712 correspond to the decrease of splenomegaly in KOKI mice. A dose of 125 mg/kg of PB is not
toxic in the transplanted model and was chosen for further experiments. Data represent
713 mean±SEM, *p<0.05; nonparametric Mann-Whitney test. KOKI+vehicle (n=3), KOKI+80

714 mg/kg PB (n=3), KOKI+100 mg/kg PB (n=3), KOKI+125 mg/kg PB (n=4).

<table><thead><tr><th>Patient N# (Polycythemia vera or Essential thrombocythemia)</th><th>JAK2<sup>V617F</sup> VAF (%)</th><th>Additional mutations (VAF %)</th></tr></thead><tbody><tr><td>1 (PV)</td><td>4.4*</td><td>nd</td></tr><tr><td>2 (PV)</td><td>12</td><td>TET2<sup>M508fs*</sup> (23)<br>BCORL1<sup>P810S</sup> (13)<br>TP53<sup>Y220C</sup> (4.5)</td></tr><tr><td>3 (ET)</td><td>8</td><td>SF3B1<sup>K666N</sup> (25)</td></tr><tr><td>4 (ET)</td><td>11</td><td>DNMT3A<sup>P700L</sup> (9)</td></tr><tr><td>5 (ET)</td><td>40.5</td><td>TET2<sup>R1452*</sup> (1.5)<br>SF3B1<sup>K666N</sup> (1)</td></tr></tbody></table>

Supplementary table 1

715

716

717

ST1. Polycythemia vera (PV) and Essential Thrombocythemia (ET) patients.

718

List of PV and ET patients, their Jak2<sup>V617F</sup> allelic frequency (VAF) and associated additional mutations. *VAF determined by PCR, the other VAFs were determined by NGS. nd: not done.

719

720

721