bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia 2 3 Gerard Minuesa1, Steven K. Albanese2,3, Arthur Chow1, Alexandra Schurer1, Sun-Mi Park1, 4 Christina Z. Rotsides4, James Taggart1, Andrea Rizzi3,5, Levi N. Naden3, Timothy Chou1, Saroj 5 Gourkanti1, Daniel Cappel6, Maria C. Passarelli3,7, Lauren Fairchild3,8, Carolina Adura9, J Fraser 6 Glickman9, Jessica Schulman10, Christopher Famulare11, Minal Patel11, Joseph K. Eibl12, Gregory 7 M. Ross12, Derek Tan4, Christina Leslie3, Thijs Beuming13, Yehuda Goldgur14, John D. Chodera3, 8 Michael G. Kharas1,* 9 10 1. Molecular Pharmacology Program, Experimental Therapeutics Center and Center for Stem 11 Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA 12 2. Louis V. Gerstner Jr. Graudate School of Biomedical Sciences, Memorial Sloan Kettering 13 Cancer Center, New York, NY, 10065, USA 14 3. Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan 15 Kettering Cancer Center, New York, NY, 10065, USA 16 4. Chemical Biology Program and Tri-Institutional Research Program, Memorial Sloan Kettering 17 Cancer Center, 1275 York Avenue, Box 422, New York, New York, NY, 10065, USA 18 5. Tri-Institutional Training Program in Computational Biology and Medicine, New York, NY, 19 10065, USA 20 6. Schrödinger GmbH, Q7, 23, 68161 Mannheim, Germany 21 7. Weill Cornell Medical College, Rockefeller University & Sloan Kettering Institute Tri-Institutional 22 MD-PhD Program, New York, NY, 10065, USA 23 8. Tri-Institutional Training Program in Computational Biology and Medicine, Weill Cornell Medical 24 College, New York, New York 10065, USA. 25 9. High-Throughput Screening Resource Center, The Rockefeller University, New York, NY, 26 10065, USA 27 10. Hematologic Oncology Tissue Bank, Department of Medicine, Memorial Sloan Kettering 28 Cancer Center, New York, New York, USA 29 11. Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, 30 NY, 10065, USA 31 12. Northern Ontario School of Medicine, Sudbury, ON, Canada 32 13. Schrödinger, Inc., 120 West 45th Street , New York , New York, NY, 10036, USA 33 14. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, 34 USA 35 36 *To whom correspondence should be addressed ([email protected]).

1 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

37 SUMMARY 38 The MUSASHI family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of 39 cancers including acute myeloid leukemia. We found that the small molecule Ro 08-2750 (Ro) 40 directly binds to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in 41 mouse and human myeloid leukemia cells resulted in an increase in differentiation and apoptosis, 42 inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA 43 depletion of MSI2. Ro demonstrated in vivo inhibition of c-MYC and reduced disease burden in a 44 murine AML leukemia model. Thus, we have identified a small molecule that targets MSI's 45 oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

2 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

46 INTRODUCTION 47 RNA-binding proteins (RBPs) play critical roles in cell homeostasis by controlling gene expression 48 post-transcriptionally. Ribonucleoprotein complexes are essential for all steps of mRNA 49 processing including splicing, polyadenylation, localization, stability, export and translation1. The 50 contribution of RBPs to tumorigenesis (e.g. SRSF2, SF3B1, MSI and SYNCRIP), through genetic 51 perturbation or epigenetic dysregulation, has been found in a variety of human cancers2-9. 52 Deregulation of the MSI family of RBPs was initially reported in gliomas10, medulloblastomas11 53 and hepatomas12. Since then, studies in a diverse range of neoplasms have involved MSI family, 54 including aggressive forms of colorectal13,14, breast15,16, lung17, glioblastoma18 and pancreatic 55 cancers19,20 and hematological malignancies. Among these tissues, the hematopoietic system 56 has been the most well characterized to dissect MSI function. The MSI2 gene was initially 57 reported as a translocation partner with HOXA9 in patients progressing from chronic 58 myelogenous leukemia to blast crisis (CML-BC)21. More recently, other rare genetic alterations 59 (involving MSI2, EVI1, TTC40 and PAX5 genes) have been identified in leukemia patients22,23,24. 60 MSI2 expression is detected in 70% of AML patients and it correlates with a poor clinical 61 prognosis in multiple hematological malignancies25-29. Thus, MSI2 has been proposed as a 62 putative biomarker for diagnosis as well as a potential therapeutic target for AML29. 63 64 The relevance and requirement of MSI2 function in leukemia was demonstrated by deletion or 65 depletion of MSI2 with a germline gene-trap knockout or shRNAs resulting in reduced 66 leukemogenesis in a CML-BC model25,26, whereas forced overexpression of MSI2 and BCR-ABL 67 or NUP98-HOXA13 leads to a more aggressive form of CML26 or myelodysplastic syndromes28, 68 respectively. MSI2 is upregulated 10-fold as CML progresses to blast crisis state in patients and 69 shRNA-mediated MSI2 silencing blocks propagation of both CML-BC and AML cell lines25,26. 70 Additionally, Msi2 was shown to be required for leukemic stem cells (LSC) in a retroviral 71 transplantation MLL-AF9 model of AML8,30. We and others have found that MSI mediates its 72 function as an RNA binding protein controling translation of its target RNAs8,25,30-32. 73 74 Overall, MSI’s requirement in myeloid leukemia makes it an attractive therapeutic target in 75 leukemia and in other malignancies33. RNA-binding proteins are often considered "undrugabble" 76 targets due to their lack of well-defined binding pockets for RNA and their absence of enzymatic 77 activity. Structurally, the MSI family of RBPs -comprising the MSI1 and MSI2- contain two highly 78 conserved RNA-recognition motifs (RRMs) in the N-terminal region and a Poly-A Binding Domain 79 (PABP) at the C-terminal region34. It is known that RRM1 is the determinant for RNA binding 80 specificity whereas RRM2, mainly adds affinity35. The minimal binding consensus described for 81 RRM1 mouse MSI1 is r(GUAG)36 and it is known that MSI it also preferentially binds UAG- 82 containing sequences in human and Drosophila35,37. Here, we describe the identification and

3 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

83 characterization of Ro 08-2750. Using biochemical and structural approaches, we find that Ro 84 binds to the MSI2 RRM1 RNA-binding site and inhibits MSI RNA-binding activity and regulation of 85 downstream oncogenic targets. Furthermore, we demonstrate that Ro 08-2750 has efficacy in 86 inhibiting leukemogenesis by in vitro and in vivo models of myeloid leukemia.

4 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

87 RESULTS 88 89 Ro 08-2750 (Ro) binds to MSI2 and inhibits its RNA-binding activity 90 In order to identify a putative MSI inhibitor, we previously performed a fluorescence polarization 91 (FP)-based screen using recombinant MSI1 and MSI2 and a consensus target RNA with a library 92 of 6,208 compounds38. We selected Ro 08-2750 (Ro) based on its RNA-binding inhibition of both 38 93 MSI1 and MSI2 . MSI2 RNA-binding inhibition was confirmed by FP (IC50 of 2.7 ± 0.4 μM) (Fig. 94 1a) We then used a chemiluminescent Electrophoresis Mobility Shift Assay (EMSA) to quantify 95 MSI2-RNA complexes in vitro. GST-MSI2 bound a MSI RNA, which was competed with the 96 addition of unlabeled RNA and by increasing concentrations of Ro (Fig. 1b and 1c). To confirm 97 the direct interaction of Ro with MSI2 protein, we performed Microscale Thermophoresis (MST)

98 assays with GST-MSI2 and found that the small-molecule interacted with a KD of 12.3 ± 0.5 μM 99 (Fig. 1d). RNA-recognition motif 1 (RRM1) of MSI2 also interacted with a similar affinity 100 (Extended Data Fig. 1a) suggesting that the binding was localized to this domain. Similarly, 101 when incubated in the presence of GST-MSI2 and a MSI RNA oligo, Ro could still compete with

102 RNA and bind to MSI2 with a KD of 27.5 ± 2.6 μM. We recently found that SYNCRIP, another 103 RNA binding protein, shares MSI2 target RNAs and is also required in leukemia4. SYNCRIP has 104 RRMs that are evolutionarily related to MSI's (with RRM1 and RRM2 sharing 33% and 57% of the 105 residues involved in RNA-binding with MSI2’s RRM1 and 2, respectively) (Extended Data Fig.

106 1b) Ro showed a 19.2-fold lower KD for SYNCRIP than for MSI2 (236.0 ± 167.1 μM, Fig.1d), 107 indicating selectivity toward MSI2. 108 109 Ro interacts with the RNA recognition site of MSI2 RRM1 110 To study how Ro interacts with the MSI2 protein, we obtained the crystal structure of apo human 111 MSI2 RRM1 at 1.7Å resolution (Extended Data Table 1, RCSB PDB accession code 6DBP). 112 This structure allowed us to perform docking analysis to identify a putative binding mode (Fig. 2a, 113 b and Extended Data Fig. 2a). Based on Ro’s ability to compete for MSI-RNA complexes, we 114 hypothesized that the binding site is likely to be shared with the RNA binding site. A closer look at 115 the residues involved in putative Ro binding interactions revealed F66 and R100 as crucial amino 116 acids participating in a stacking interaction with the planar tricyclic structure of the small-molecule 117 (Fig. 2b). Also, the NH backbone group from F97 formed a stabilizing H-bonding with the oxygen 118 from the aldehyde moiety (Fig. 2b, c). A 2D representation of the interacting partners showed 119 R100 forming a π-cation interaction and K22 as a putative amino acid forming an H-bonding with 120 the opposite ring of Ro structure (Fig. 2c). To confirm these putative interactions, we performed 121 site-directed mutagenesis on the full-length MSI2 protein by mutating the F97 or the main three 122 potential residues involved in Ro binding (F66, F97 and R100) to alanine. MST interaction assays

123 showed a nearly 7-fold decrease in affinity (measured KD 69.5 ± 14.7 for F97A versus 10.5 ± 0.3 124 μM for wild-type) for the single mutant. More dramatically, the triple mutant (F66/F97/R100) was

5 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

125 incapable of binding Ro, confirming our hypothesis that Ro binds at the RNA-interacting site and 126 can compete for it. We further validated if the mutations of these three residues also disrupt RNA 127 binding by MST: wild-type and F97A possess equivalent RNA binding affinities, whereas the triple

128 mutant F66/F97/R100 only partially ablated RNA binding (KD > 50 μM), (Extended Data Fig. 2b). 129 130 To further test structure activity relationships, we obtained two Ro related molecules (Ro-NGF 131 and Ro-OH). The first analog, Ro-NGF, was selected to determine if Ro’s activity was related to 132 its anti-NGF activity, as previously described39 because this compound showed the highest

133 affinity (KD [NGF] = 1.7 μM) for NGF in its compound series (Extended Data Table 2). The second 134 analog, Ro-OH, a reduced form of the aldehyde to an alcohol, contained a single alteration to the 135 Ro aldehyde moiety (Fig. 2e and Extended Data Figs. 2c, 3a, b). Alchemical free energy

136 calculations showed computed binding free energies (ΔGbind) for the three ligands (Ro, Ro-OH 137 and Ro-NGF) in a similar range, with a slightly higher affinity predicted binding for Ro and Ro-OH 138 (-5.5 and -6.1 vs -5.1 kcal/mol for Ro-NGF) (Extended Data Fig. 4a). Both MSI2 protein and 139 ligands adopted a conformationally heterogeneous ensemble of binding poses, with the protein- 140 ligand complex predicted to undergo a slight conformational change for Ro and Ro-OH upon 141 binding (Extended Data Fig. 4b). Free energy calculations for all three small-molecules suggest 142 that Ro-NGF adopts a much more diverse set of conformations (as measured by conformational 143 clustering of the fully-interacting alchemical state) than Ro-OH or Ro (Fig. 2f). Ro showed the 144 fewest clusters, with the top three clusters accounting for 92.7% of the sampled configurations 145 (Extended Data Fig. 4c). Ro-OH showed a larger number of clusters, with the four clusters 146 accounting for 49.1% of sampled configurations, indicating a greater degree of heterogeneity than 147 Ro (Extended Data Fig. 4d). Ro-NGF displayed an even greater degree of heterogeneity, 148 showing a large number of low population of clusters (data not shown). Further structural analysis 149 of our docked model suggests Ro-OH lacking the R100 π-cation interaction and Ro-NGF in a 150 displaced position from the RNA-binding core (Extended Data Fig. 2e, f, g, h) as compared to 151 Ro, despite similar interacting residues. To experimentally validate these predictions, we 152 performed EMSA of GST-MSI2 competing Ro-OH and Ro-NGF with RNA, comparing potency 153 with Ro and unlabeled RNA as positive controls. Accordingly, whereas Ro-OH showed partial 154 (∼30-40%) but significantly poorer inhibition than Ro (65-75%, p<0.05), Ro-NGF showed no 155 ability to displace MSI2-RNA complexes (Figure 2g and 2h). These results were further 156 confirmed by FP assay with Ro-OH inhibiting with 12.5-fold less potency than Ro, and Ro-NGF 157 failing to inhibit of RNA-binding activity (Supplemental Figure 2d). Furthermore, in MST assays,

158 Ro-OH showed a 27-fold lower affinity than Ro (KD 302.0 ± 119 μM for Ro-OH versus 11.2 ± 0.6 159 μM for Ro) for GST-MSI2, whereas Ro-NGF failed to demonstrate any interaction (Figure 2i). 160 Thus, our structural and biochemical experimental data support the conclusion that Ro and MSI2

6 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

161 interact via the RRM/RNA binding site and that the drug can displace RNA from its binding site, 162 thus likely inhibiting MSI-related translational regulation. 163 164 Ro 08-2750 demonstrates therapeutic efficacy in murine MLL-AF9 leukemic cells 165 To test the MSI-inhibitory effect of Ro in a murine AML of leukemia, we used MLL-AF9 166 expressing leukemic BM cells from secondary transplants previously established in the lab8. We 167 first assessed the cytotoxicity effects of Ro and the two analogs against these leukemia cells. 168 Consistent with an on-target effect on MSI inhibition and in agreement with the RNA-binding 169 activity inhibition assays, Ro effectively inhibited leukemia cell proliferation (half-effective

170 concentration, EC50 = 2.6 ± 0.1 μM). By comparison, the analogues that failed to interact with

171 MSI2 had a diminished effect (Ro-OH EC50 = 21.5 ± 0.8 μM; Ro-NGF > 50 μM), suggesting that 172 the antiproliferative effect is due to the ability of Ro to inhibit MSI2 RNA binding-activity (Fig. 3a). 173 Treatment of cells with Ro resulted in an increase in the myeloid and granulocyte markers (Mac1 174 and Gr1, respectively) at 5 μM dose and 48h treatment as seen by both flow cytometry (Fig.3b) 175 and morphologically by Eosin Y and Methylene Blue/ Azure A staining (Fig. 3c). When we 176 assessed apoptosis at different time points, we found a significant increase in the Annexin V+ 177 population as early as 8h (both at 5 and 10 μM) with the highest increase at 48h and 10 μM Ro 178 (Fig. 3d and Extended Data Fig. 5). 179 180 We then assessed how MSI2 overexpression affected the plating capacity of MLL-AF9 BM cells 181 in culture in the absence or presence of Ro. MSI2 overexpressing cells formed 50% more 182 colonies than control cells transduced with an empty vector (MIB). Treatment of cells with Ro 183 resulted in reduced colony formation in control cells by >50% and ∼75% at 1 μM and 5 μM 184 concentrations, respectively. MSI2-overexpressing leukemia cells however showed increased 185 resistance to these doses (Fig. 3e). Of note, we assessed MSI2 translational targets8,31 in these 186 cells by immunoblotting and we found that Ro treatment reduced protein abundance of SMAD3, 187 c-MYC and HOXA9 in control cells, whereas the levels of these proteins remained unaffected in 188 cells that overexpressed MSI2 (Fig. 3f). Indicating a potential therapeutic window between 189 normal and malignant cells, Ro abolished MLL-AF9+ BM colony formation at concentrations that 190 did not affect the plating efficiency of normal Lin-Sca+cKit+ (LSK) cells (Fig. 3g),. 191 192 Ro 08-2750 treatment inhibits survival of human AML cell lines and patient cells 193 To determine if Ro also has activity against human myeloid leukemia, we first tested cytotoxicity 194 effects of the small-molecule and the two analogs in MOLM13 (AML, MLL-AF9+) and K562 195 (CML-BC, BCR-ABL+) cell lines, both known to require MSI2 function4,26. Consistent with our 196 previous data in MLL-AF9 cells, we observed that in both these leukemia cell lines, Ro

197 demonstrated anti-proliferative effect (EC50 ∼8 μM), whereas the two analogs (Ro-OH and Ro-

7 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

198 NGF) revealed a >4.5-fold weaker potency. Ro induced myeloid differentiation and apoptosis in 199 both K562 and MOLM13 cells based on flow cytometry and by morphology (Fig. 4b-d and 200 Extended Data Fig. 6a-c and 6b). Plating activity was >80% inhibited at the 20 μM Ro dose in 201 the human AML cell lines (Fig. 4e). Additionally, Ro demonstrated differential sensitivity in three 202 AML patient samples (Extended Data Table 3) colony plating assays compared to normal 203 human CD34+ cord blood cell (>50% inhibition in colony numbers at 5 μM comared to only a 204 modest reduction at 20 μM Ro, Fig. 4f). These results indicate that Ro can induce differentiation

205 and apoptosis in primary human AML cells and spare normal stem cells up to 2 x EC50 Ro. 206 207 Ro 08-2750 inhibits binding of MSI2 to its RNA targets and exhibits gene signature from 208 MSI2 depleted cells 209 To further investigate the effect and mechanism of action of Ro, we initially performed RNA 210 immunoprecipitation (RNA-IP with FLAG) experiments on K562-MIG (empty vector) and K562-

211 FLAG-MSI2 (MSI2 overexpressing) cells (Fig. 5a). After incubating the drug at 10 μM (∼EC50) for 212 1 hour with the cells, we could detect a significant decrease in MSI2 mRNA binding targets 213 (TGFBR1, cMYC, SMAD3, CDKN1A) (Fig. 5b). These data suggest that Ro can block MSI2 214 binding to target mRNAs in a cellular context at a short time-point. 215 216 To globally assess the proximal effect of Ro treatment on the transcriptional program, we then 217 performed RNA-sequencing on MOLM13 and K562 cells after 4 hours of treatment. Ro incubation 218 resulted in modest but significant gene expression changes in both the MOLM13 and K562 AML 219 cells (59 upregulated, 221 downregulated and 111 upregulated, 164 downregulated, respectively; 220 FDR<0.05), (Extended Data Tables 4-5). Most importantly, this Ro signature enriched for the 221 gene expression profiling after shRNA mediated depletion of MSI2 in CML-BC (AR-230 and 222 LAMA84) and AML cell lines (THP1 and NOMO-1) (Fig. 5c)26. To annotate the functional 223 pathway overlap with Ro treatment in both cell lines and MSI2 shRNA depletion, we performed 224 gene-set enrichment analysis (GSEA)40 on all 4,733 curated gene sets in the Molecular 225 Signatures Database (MSigDB, http://www.broadinstitute.org/msigdb) combined with 92 226 additional relevant gene sets from our experimentally derived or published hematopoietic self- 227 renewal and differentiation signatures31,40. Interestingly, we observed an overlap of MSI- 228 associated signatures from our previous dataset and an enrichment with MSI1 direct mRNA 229 targets from the intestine (Extended Data Tables 7-12 and Extended Data Fig. 7a)4. Moreover, 230 we observed a ~70% overlap of the functional pathways between each individual cell line and the 231 pathways altered after shRNA depletion of MSI2 (Fig. 5d). Among these shared pathways, 76% 232 (543 out of 717) overlapped in MOLM13 compared to K562 cells treated with Ro, which included 233 c-MYC, mRNA related and leukemia associated gene sets (Fig. 5d and Extended Data Table

8 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

234 12). Thus, Ro treatment after a short administration recapitulated a large portion of the MSI2- 235 associated gene expression program. 236 237 To determine how Ro affects previously determined MSI targets, we treated both K562 and 238 MOLM13 cells with increasing concentrations of Ro (up to 20 μM at 4 hours). In previous studies, 239 MSI was demonstrated to maintain the protein levels of TGFβR1, c-MYC, SMAD3 and HOXA98,31 240 while suppressing P21 abundance41,42. Consistent with this, we observed a significant and dose 241 dependent reduction of TGFβR1, c-MYC, SMAD3, HOXA9 and an increase in the protein 242 abundance of P21, while the non-target control β-ACTIN remained unchanged (Fig. 5d and 5e). 243 Additionally, Ro could inhibit MSI2 targets in a time-dependent manner with c-MYC, a short half- 244 life protein, being reduced in 1 hour of treatment (Fig. 5f and 5g). In support of Ro altering 245 translation of specific MSI2 targets but not generally inhibiting global translation, we found 246 equivalent global protein synthesis after drug treatment as assessed by O-propargyl-puromycin 247 incorporation (Extended Data Fig. 7b). As previously noted by RNA-sequencing, there were 248 modest effects on the mRNA expression of MSI2 targets by qPCR (Extended Data Fig. 7c) 249 suggesting that Ro mainly influences its direct targets through a post-transcriptional mechanism. 250 Thus, these results support our hypothesis that Ro acts in the MSI-related translational program. 251 252 Ro 08-2750 inhibits leukemogenesis in an in vivo MLL-AF9 model of myeloid leukemia 253 Finally, we sought to determine if Ro has activity in vivo using an aggressive murine MLL-AF9 254 murine leukemia model. Acute treatment of Ro (4h and 12hr) reduced c-KIT protein abundance 255 and intracellular c-MYC (Fig. 6a-c). To determine if Ro treament could affect disease burden we 256 next treated a second cohort of animals and monitored them for disease progression for 19 days 257 after transplantation (Fig. 6d). Ro administration every 3 days was well tolerated (Extended Data 258 Fig. 7a, b, c) demonstrating little to no weight loss and equivalent red blood cells and platelets 259 counts compared to control group. When control mice succumbed to disease (day 19 post- 260 transplantation), we assessed the disease in both groups and found a significant reduction in 261 spleen weights (Fig. 6e), white blood cell counts (Fig. 6f) and c-MYC levels compared to the 262 controls (Figure 6g). These data provide the feasibility that targeting MSI in vivo could have 263 therapeutic efficacy in AML.

9 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

264 DISCUSSION 265 Inhibiting MSI RNA-binding activity could represent a novel therapeutic avenue in both 266 hematological malignancies and solid cancers. Our previous FP-based screen identified 267 compounds that inhibit MSI binding to RNA38. Here, we characterize Ro 08-2750 as a first 268 selective MSI inhibitor with biochemical, structural and cellular validation linking the compound to 269 the inhibition of the MSI program. Ro falls in the low micromolar range of activity, in line with other 270 RBP associated inhibitors43-47. We validated Ro as a MSI2 RNA-binding inhibitor with biophysical 271 and biochemical assays by utilizing a high-resolution crystal structure of the MSI2 RRM1. Our 272 newly developed computational molecular modeling algorithm and docking analysis, allowed to 273 predict and validate the key MSI2 residues that were critical for the interactions with Ro in the 274 RNA binding site. Both our novel crystal structure and the computational tools will be useful for 275 the discovery and development of small-molecule RBPs inhibitors. We found that a single 276 chemical reduction of Ro decreased its activity in both in biochemical and in vitro cell based 277 assays. Utilizing a related compound with high affinity binding to NGF, we found that it no longer 278 bound MSI2 and poorly inhibited leukemia cell growth. Further studies involving medicinal 279 chemistry with heterocycle isoalloxazines or pteridine-derived compounds could help identifying 280 more selective and potent MSI-inhibitors. 281 282 Other groups have identified agents that have putative MSI1 inhibitory activity. A natural phenol 283 extracted from cottonseed ((-)-gossypol) was shown to reduce MSI1 to bind RNA45 but this 284 interaction was not validated by structure-activity relationships. Of note, (-)-gossypol has been 285 considered to be a pan-active compound that has hit in multiple HTS screens48-50 and assigned to 286 have activity against Bcl-251. MSI1 activity was also inhibited by ω-9 monounsaturated fatty acids 287 (e.g. oleic acid), allosterically binding and inducing a conformational change that prevents RNA to 288 bind52. It remains unclear if (-)-gossypol or oleic acid have a more broad RNA binding protein 289 inhibitor activity as they were not directly tested against any other RBPs43,45,52. We found that Ro 290 could demonstrate differential binding activity to MSI2 compared to SYNCRIP, Ro’s effect on 291 colony formation and direct targets could be rescued by MSI2 overexpression. Moreover, we 292 observed a strong enrichment for the MSI2 shRNAs gene expression signature, associated 293 functional pathways, inhibition of MSI2 binding of target mRNAs and reduced abundance of 294 MSI2 direct targets after Ro treatment. In contast to other general translational inhibitors Ro did 295 not alter global translation53,54. These data suggest that Ro could be used to probe the acute 296 effects of MSI inhibition in a variety of cellular contexts and cancer models. 297 298 It is also important to note that Ro inhibits both MSI1 and MSI2 and although MSI1 is expressed 299 at low levels in myeloid leukemia it could still be blocking residual MSI1 activity. Moreover, in 300 other models such as the intestine where both factors act redundantly13, dual inhibition could

10 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

301 provide a powerful therapeutic strategy. Of note based on the close conservation of the RRMs 302 between the two proteins it might be challenging to design MSI1 or MSI2 selective inhibitors. 303 304 We demonstrated a therapeutic index for Ro in human AML patient samples versus cord-blood 305 derived CD34+ human stem and progenitor cells. Despite the challenges for in vivo 306 administration, we reduced the disease burden in an aggressive MLL-AF9 leukemia model and 307 decreased MYC levels without overt toxicity. Interestingly, it has previously been shown that MSI2 308 can contribute to chemotherapeutic resistance in different cancer models42,55,56. Future studies 309 could examine if combination therapies could provide additional clinical benefit. 310 311 This study identifies and characterizes Ro 08-2750 as the first compound selectively inhibiting the 312 oncogenic RNA-binding activity of MSI in myeloid leukemia. It will be important to use this 313 compound (or other chemical derivatives) to test their efficacy in other cancer models and on MSI 314 function related to normal physiology. We suggest that Ro provides the rationale for developing 315 more potent compounds with improved clinical utility for the treatment of cancers that are 316 dependent on the MSI family. Additionally, as there are hundrends of RRM containing RNA 317 binding proteins, Ro targeting an RRM motif to block RNA activity represents a valuable proof of 318 concept for the general inhibition of these class of RNA regulators. Thus, we provide a framework 319 to identify and test novel RNA binding protein inhibitors in cancer. 320

11 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

321 Methods 322 Purification and culture of cord blood derived HSPC-CD34+ cells 323 Mononuclear cells were isolated from cord blood using Hetarstach solution (6% Hetastarch in 324 0.9% NaCl) and Ficoll-Hypaque Plus density centrifugation. CD34+ Hematopoietic Stem and 325 Progenitor Cells (HSPCs) were subsequently purified by positive selection using the Auto MACS 326 Pro Separator and isolation kit (Miltenyi) and were cultured in Iscove's modified Dulbecco's 327 medium (IMDM, Cellgro), 20% BIT 9500 medium (Stem Cell Technologies) supplemented with 328 SCF (100 ng/ml), FLT-3 ligand (10 ng/ml), IL-6 (20 ng/ml) and TPO (100 ng/ml) as the basic 329 culture. All cytokines were purchased from Peprotech, NJ. 330 331 Isolation and viral transduction of murine MLL-AF9 leukemia and normal cells 332 Tibia, femurs, pelvis, and arm bones from leukemia or C57BL/6 wild type mice (10-12 weeks old) 333 were harvested, crushed, filtered, and subjected to red blood cell lysis (Qiagen). To isolate c-Kit+ 334 cells, bone marrow cells were incubated with anti-CD117 microbeads (Miltenyi Biotec), according 335 to manufacturer’s instructions, and then subjected to positive selection using autoMACS Pro 336 Separator. For MLL-AF9 BM cells, previously thawed vials from secondary transplants (Park et al. 337 2015) were used. All murine cells were cultured and transduced in RPMI with 10% FBS and 338 cytokines SCF (10 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml) and GM-CSF (10 ng/ml). For MSI2 339 overexpression, cells were spinfected with viral supernatant containing MSCV-IRES-BFP or 340 MSI2-IRES-BFP contructs (see Cloning section). 341 342 Colony forming unit (CFU) assays 343 10,000 leukemic MLL-AF9 BM cells or c-Kit enriched normal stem cells (Lin-Sca-Kit+) were 344 plated on methylcellulose-based culture media (methocult) GFM3434 (Stem Cell Technologies). 345 Colonies were scored every five days for leukemia cells and every seven to nine days for normal 346 c-kit-enriched bone marrow cells. For human cells, 5,000 of the leukemia cel lines K562 (CML- 347 BC) or MOLM13 (AML) and 10,000 of HSPCs CD34+ or AML patient cells were plated (in 348 duplicate) in methylcellulose (MethoCultTM H4434 Classic, Stem Cell Technologies). CFU 349 colonies in HSPCs CD34+ were scored 14 days after seeding. AML patient cells characteristics 350 are shown in Extended Data Table 3. 351 352 Flow cytometry 353 To monitor the differentiation status, 200K MLL-AF9 BM cells DMSO or Ro treated (during 8, 16, 354 24, 48h) were stained with the following antibodies: anti-CD11b (Mac1)-PE (clone M1/70, 355 #101208, BioLegend), anti-Ly-6G (Gr1)-APC (clone RB6-8C5, #17-5931-82, eBioscience), and 356 anti-CD117 (c-Kit)-APC-Cy7 (clone 2B8, #105826, BioLegend). For the human cell lines 357 differentiation, we used two panels: (1) anti-CD14-PE (clone M5E2, #555398, BD Pharmingen),

12 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

358 anti-CD13-APC (clone TUK1, #MHCD1305, Life Technologies); (2) anti-CD71-APC (clone 359 CY1G4, #334104, BioLegend), anti-CD235a (Glycophorin A)-PE (clone YTH89.1, #MA5-17700, 360 Invitrogen). All samples were stained for 20min in the dark, washed once with PBS 1X and re- 361 suspended in RPMI + 2% FBS for analysis. For intracellular flow cytometry detection of cMYC, 1- 362 2x106 cells were fixed in 2% paraformaldehide for 15 minutes, washed 2 times with 1X PBS and 363 permeabilized with cold methanol and kept at -80 until use. For the staining, cells were washed 364 twice in 1X PBS and stained in 100 μl final volume. c-MYC (5605, Cell Signaling Technology non- 365 labelled primary antibody was incubated at 1/200 dilution for 1h and labelled donkey anti-rabbit 366 Alexa Fluor 568 (#A10042, Invitrogen) or goat anti-rabbit Alexa Fluor 647 (#A21245, Invitrogen) 367 were used at 1/400 for 20-30 minutes. Cells were washed once with PBS 1X and re-suspended in 368 RPMI + 2% FBS for analysis. All flow cytometry analysis was performed in a LSRII or LSR 369 Fortessa (BD Biosciences) and data was graphed by using FlowJoTM version 10.4. 370 371 Morphological analysis 372 After the appropriate time of Ro treatment (or DMSO in controls) in culture, 1.5x105 MLL-AF9 BM 373 or human leukemia cells (K562 and MOLM13) were washed once with 1X PBS, counted and 374 centrifuged onto slides for 5 minutes at 500 rpm and air-dried for 24h prior to Richard-Allan 375 Scientific Three-Step Stain Staining Set (Thermo Scientific) based on Eosin Y and Methylene 376 Blue/ Azure A and mounted with Permount solution (Fisher). Cell morphology was evaluated by 377 light microscopy at 400X magnification (Zeiss Imager M-2, equipped with AxioCam ERc 5s). 378 379 Apoptosis measurements 380 Apoptosis measurements were taken by MUSETM Cell Analyzer (Millipore) using the MUSETM 381 Annexin V and Dead Cell Assay Kit (Millipore) as recommended by the instructions from the 382 manufacturer. Dot plots showing viability versus Annexin V+ cells are shown in Extended Data 383 Figures 4 and 5. 384 385 In vivo transplantation of leukemia cells and Ro 08-2750 administration 386 10,000 of MLL-AF9 BM secondary mouse leukemia cells previously obtained1 were injected retro- 387 orbitally into female C57BL/6 (10-12 weeks old) recipient mice that had been sublethally 388 irradiated at 475 cGy. Drug administration (Ro 08-2750, 13.75 mg/Kg, DMSO) was performed by 389 intraperitoneal injections (50 μL, top tolerated DMSO volume) 3 weeks after BM transplants 390 (when showing signs of disease) for pharmacodynamic experiments (see Fig. 6a), and 3 days 391 after BM transplant for in vivo long-term studies (see Figure 6d). Mice weight were monitored 392 every day to check for toxicity. All animal studies were performed on animal protocols approved 393 by the Institutional Animal Care and Use Committee (IACUC) at Memorial Sloan Kettering Cancer 394 Center.

13 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

395 396 Fluorescence Polarization (FP) to assess RNA-binding activity inhibition 397 To validate RNA-binding activity inhibition by Ro 08-2750 and derivatives (Ro-OH, Ro-NGF) we 398 used Fluorescence Polarization (FP) based assay in as previously described in 384-well format 2 399 for dose-response curve studies . As previously, the RNA oligo used (Cy3-C9-[spacer]- 400 rGUAGUAGU, Integrated IDT Technologies) contained 2 MSI motifs (GUAGU) and was 8- 401 nucleotides long, optimal to minimize background and unspecific interactions. Differently, here 402 manual pipetting was used to plate the reagents and the FP reading was performed in a BioTek 403 Synergy Neon Plate Reader (High-Throughput Screening Resource Center, Rockefeller 404 University). 405 406 Binding affinity quantifications by MicroScale Thermophoresis (MST) 407 408 For binding affinity studies of RNA and small-molecules to proteins of interest, purified 409 recombinant GST-MSI2 WT, F97A and F66A/F97A/R100A mutants and GST-SYNCRIP were 410 NT647-labeled using an amine-coupling kit (NanoTemper Technologies). Runs were performed 411 at a concentration range of 50-125 nM (MSI2 and mutants) and 60 nM (SYNCRIP) to get optimal 412 fluorescence signal using an LED power of 40-50% in a red laser equipped Monolith NT.115 413 (NanoTemper Technologies) (High-Throughput Screening Resource Center, Rockefeller 414 University). Prior to each run, protein preparations were diluted in MST buffer (50 mM HEPES, 415 100 mM NaCl, 0.05% Tween-20, pH 7.4) and aggregation was minimized by centrifuging the 416 solutions at 15,000 rpm for 10 minutes. GST-proteins or GST-protein/RNA complexes (15 min 417 pre-incubation) were mixed with increasing concentrations of small-molecules (0.015 to 500 μM) 418 or RNA (0.0015 to 50 μM) and loaded onto 16 Premium Coated capillaries. The RNA oligo used 419 (rGUAGUAGUAGUAGUA, Integrated IDT Technologies) contained 4 MSI motifs (GUAGU) and 420 was 15-nucleotides long. The MST measurements were taken at RT and a fixed IR-laser power 421 of 40% for 20 seconds per capillary. GraphPad Prism was used to fit the normalized data and

422 determine apparent KD values, represented as percent of fraction bound. 423 424 Chemiluminescent Electrophoresis Mobility Shift Assays (EMSA) 425 An EMSA approach to assess MSI2-RNA complexes and the inhibitory effect of small-molecules 426 was set up by using LightShift Chemiluminescent RNA EMSA kit (Thermo Scientific). In brief, 427 GST-MSI2 (125-250 ng) was preincubated with DMSO or the small-molecule (typically 20 μM 428 final concentration) during 1h at RT in EMSA buffer 1X RNA EMSA binding buffer (10 mM

429 HEPES, 20 mM KCl, 1 mM MgCl2, 1 mM DTT, Thermo Scientific) supplemented with 5% glycerol, 430 100 μg/mL tRNA and additional 10 mM KCl. After this period, 40 nM of biotinylated-RNA (biotin- 431 rGUAGUAGUAGUAGUA, Integrated IDT Technologies -same as for MST-) was added to the 432 mixture (20 μL final volume) and incubated another 1h at RT. During this second incubation

14 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

433 period, a 4-20% TBE polyacrylamide gel (BioRad) was pre-run at 100V for 30-45min in cold 0.5X 434 TBE (RNAse free). 5 μL of 5X loading buffer was added to the 20 μL reaction and loaded into the 435 pre-run TBE gel and voltage set at 100V. Samples were electrophoresed until 3/4 of the length of 436 the gel. Samples were then transferred in 0.5X TBE at 350-400 mA for 40 min. Membranes were 437 then crosslinked with UV-light crosslinking instrument (UV Stratagene 1800) using Auto-Cross 438 Link function. Membranes were either stored dry for development next day or developedusing the 439 detection biotin-labeled RNA chemiluminescence kit (as indicated by the manufacturer) (Thermo 440 Fisher) and Hyperfilm ECL (GE Healthcare). 441 442 Cloning, expression, and purification of GST tagged proteins 443 Human full-length MSI2 was cloned into the retroviral backbone pMSCV-IRES-BFP (MIB) vector 444 (a gift from Dario Vignali; Addgene plasmid # 52115) by Custom DNA Constructs (University 445 Heights, Ohio) introducing a 5'Flag tag and using BamHI and EcoRI restriction sites. Human full- 446 length MSI2 was previously cloned into pGEX6P3 as described2. RNA-recognition motif 1 447 (RRM1) from human MSI2 (nucleotides #64-270, NM_138962.2) was subcloned into empty 448 pGEX6P3 using EcoRI and NotI restriction sites. Human SYNCRIP (hnRNP-Q variant 3, 449 NM_001159674.1) was subcloned into empty pGEX6P3 (GE Healthcare) by introducing a 5'Flag 450 sequence (5'-ATGGATTACAAGGATGACGACGATAAG-3') and using SalI and NotI sites. GST- 451 Flag-MSI2 wild-type (WT), Flag-MSI2 mutants (F97A, F66A/F97A/R100A), GST-RRM1 and GST- 452 Flag-SYNCRIP recombinant proteins were produced in BL21 (DE3) competent cells (Agilent 453 Technologies, Santa Clara, CA) as previously reported for MSI2 WT2. Here, GST-SYNCRIP 454 protein needed higher content of NaCl (250 mM) in the 1X PBS dialysis step and final buffer for 455 optimal storage and performance in the biochemical and biophysical assays performed. 456 457 Site-directed mutagenesis 458 To perform site-directed mutagenesis into pGEX6P3-Flag-MSI2 construct and express the 459 corresponding recombinant GST-MSI2 mutants, we used QuikChange Lightning and Multi Site- 460 Directed Mutagenesis Kit (#210513 and #210518, Agilent Technologies). The primers were 461 designed using QuickChange Primer Design 462 (https://www.genomics.agilent.com/primerDesignProgram.jsp) and were the following: F66A 463 (Fwd: 5'-GCTCCAGAGGCTTCGGTGCCGTCACGTTCGCAG-3', Rev: 5'- 464 CTGCGAACGTGACGGCACCGAAGCCTCTGGAGC-3'; F97A/ R100A (Fwd: 5'- 465 AGACGATTGACCCCAAAGTTGCAGCTCCTCGTGCAGCGCAACCCAA-3', Rev: 5'- 466 TTGGGTTGCGCTGCACGAGGAGCTGCAACTTTGGGGTCAATCGTCT-3') and R100A (using 467 F97A mutant construct as template) (Fwd: 5'- 468 CCAAAGTTGCAGCTCCTCGTGCAGCGCAACCCA-3', Rev: 5'-

15 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

469 TGGGTTGCGCTGCACGAGGAGCTGCAACTTTGG-3'). PCR reactions and cloning were 470 perfomed as indicated by the manufacturer (Agilent Technologies). 471 472 Human MSI2 RRM1 recombinant protein production 473 GST-RRM1 protein was initially produced in BL21 (DE3) competent cells (Agilent Technologies, 474 Santa Clara, CA) as previously reported for MSI2 WT2. Here, the cell lysate of 4L initial culture 475 was centrifuged at 15,000 rpm for 1h and the resulting volume applied to a XK16/20 column pre- 476 packed with Glutathione Sepharose 4 Fast Flow connected to an AKTA Prime FPLC (GE 477 Healthcare). To obtain the RRM1 optimal prep for the crystal preparation, the collected fractions 478 containing GST-RRM1 (in 50 mM Tris-HCl, 20 mM reduced L-Glutathione) were pooled and 479 dialyzed against PreScission Protease Buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1mM 480 DTT, pH 7.5). GST tag was then cleaved with PreScission Protease overnight at 4°C. Pure RRM1 481 fractions were obtained through size exclusion chromatography (HiLoad Superdex 75, GE 482 Healthcare) and concentrated with a 3K Amicon Ultra Centricon (Millipore). 483 484 Crystallization and structure determination 485 A final concentrated MSI2 RRM1 pure protein preparation (>98% by coomassie) at 2 mg/mL in 50 486 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1mM DTT, pH 7.5 was crystallized by sitting drop 487 vapor diffusion. A 1 uL of protein solution was mixed with an equal volume of precipitant solution 488 containing 100 mM Tris, 200 mM Li2SO4, 25% PEG (pH 8.5). Crystals appeared after two weeks. 489 They were cryoprotected by mother liquor containing 25% glycerol and flash frozen in liquid 490 nitrogen. X-ray diffraction data were collected from single crystals at the Advanced Photon 491 Source beamline 24ID-C at 100 K. Indexing and merging of the diffraction data were performed in 492 HKL20003. The phases were obtained by molecular replacement by PHENIX4 using PDB entry 493 1UAW as the search model. Interactive model building was performed using O5. Refinement was 494 accomplished with PHENIX. Data collection and refinement statistics are summarized in 495 Extended Data Table 1. The crystal structure has been deposited in RCSB PDB under the 496 accession code 6DBP. 497 498 RNA purification and quantitative real-time PCR 499 Total RNA was isolated from 1-2x106 cells dry pellets kept at -80C for less than a week using 500 Qiagen RNeasy Plus Mini kit. cDNA was generated from RNA using iScript cDNA Synthesis 501 (#1708891, BioRad) with random hexamers according to the manufacturer's instructions. Real- 502 time PCR reactions were performed using a Vii7 sequence detection system. β-ACTIN was 503 commonly used to normalize for cDNA loading. Relative quantification of the genes was 504 performed using Power SYBR Mix (2X) and specific primers for c-MYC, TGFβR1, SMAD3, ΔΔ 505 HOXA9 and CDKN1A and the 2− Ct method as described by the manufacturer.

16 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

506 507 Immunoblot analysis 508 For immunoblot analysis, Ro treated and DMSO control MOLM13 or K562 cells (routinely at 509 0.5x106 cells/ mL) were counted and washed twice with cold PBS before collection. 1-5x106 cells 510 were resuspended and lysed in 250 μl of 1X RIPA Buffer supplemented with Protease Inhibitor 511 Tablets (Sigma-Aldrich) buffer for 30min on ice. After centrifugation at 14,000rpm on a top-bench 512 centrifuge, lysate (supernatant) was collected and total protein quantified by BCA (Thermo 513 Scientific). Cell lysates were separated by 4–15% SDS–PAGE and transferred to 0.45 μm 514 nitrocellulose membrane. Membranes were blocked and were blotted overnight (4C) for TGβR1 515 (ab31013, Abcam, 1:750 dilution), SMAD3 (9523S, Cell Signaling Technology, 1:750 dilution), 516 HOXA9 (07-178, Millipore, for drug dose-dependent experiments and ab140631, Abcam; 1:1,000 517 dilution for time-course experiments), c-MYC (5605, Cell Signaling Technology; 1:1,000 dilution), 518 P21 (2947S, Cell Signaling Technology, 1:750 dilution), MSI2 (ab76148, Abcam; 1:2,000 dilution) 519 and β-ACTIN-HRP conjugated (A3854, Sigma-Aldrich; 1:20,000 dilution) and developed by 520 Hyperfilm ECL (GE Healthcare) with ECL and pico-ECL reagents (Thermo Scientific). 521

522 Luminescence-based cytotoxicity assays (EC50) 523 10,000 cells (MLL-AF9 BM from secondary transplants or human leukemic cell lines -K562 or 524 MOLM13-) were platted into U-bottom 96-well plates in the presence of increasing concentration 525 of small-molecules (Ro, Ro-OH or Ro-NGF) up to 100 μM (in 1:2 serial dilutions). Cells were TM 526 cultured for 72h at 37C in a 5% CO2 incubator. To read cell viability, Cell-Titer Glo kit 527 (Promega) was used. After cooling down cells to RT for 20-30min, 100 μL of the cultured cells 528 were transferred to opaque-white bottom 96-well plates and mixed with 100 μL of Cell-Titer GloTM 529 Reagent (previously prepared by mixing buffer and powdered substrate). The mixture was 530 incubated for 15min at RT and read using a Synergy H1 Hybrid reader (BioTek) for 531 luminescence. Data was normalized as percentage viability and graphed by non-linear regression 532 curves in Graph Pad PRISM 7.0. K562 and MOLM13 lines were purchased from ATCC, 533 authenticated by Genetica, and tested negative for mycoplasma contamination. 534 535 RNA immunoprecipitation (RNA-IP) 536 To assess mRNA enrichment and blocking of protein-binding to mRNA by the small-molecules 537 we performed RNA immunoprecipitation (RNA IP) experiments using Magna RIP RNA-binding 538 protein immunoprecipitation kit (#03-115, Millipore). 25 × 106 K562-MIG or MSI2 overexpressing 539 cells 1h treated with DMSO (control) or Ro μM were used. First, cells were washed with cold PBS 540 and lysed. Five micrograms of mouse anti-Flag (clone M2, #F1804, Sigma-Aldrich) antibody 541 incubated with magnetic beads were used to immunoprecipitate Flag-MSI2 K562 cells. After 542 washing the immunoprecipitated, they were treated with proteinase K. RNA extraction was

17 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

543 performed by the phenol–chloroform method, and 200-500 ng of purified RNA was converted to 544 cDNA using the Verso cDNA kit (Thermo Scientific). qPCR was used to validate target mRNAs 545 bound by MSI2 and control cells. 546 547 O-Propargyl-Puromycin incorporation by flow cytometry 548 Cells were plated at a density of 200,000 cells/ml and pre-treated with DMSO or Ro up to 4h. 549 Then, 50 μM O-propargyl-puromycin (OP-Puro; NU-931-05, Jena Bioscience) was added. Control 550 cells were co-incubated with DMSO or Ro and treated with 150 μg/ml cycloheximide for 15 min. 551 Non-OP-Puro treated cells were also used as negative controls for flow cytometry. Cells were 552 washed twice before collection and subjected to processing using the Click-iT Flow Cytometry 553 Assay kit (C10418, Invitrogen) following the manufacturer's instructions. Labeled cells were 554 analyzed using a BD LSR Fortessa instrument and graphed as Alexa Fluor 647 (AF647) Mean 555 Fluorescence Intensity (normalized to DMSO control treated with OP-Puro). 556 557 RNA sequencing 558 Total RNA was isolated from 1x106 dry pellets of K562 and MOLM13 4h treated with DMSO 559 (control) or Ro 20 μM (n = 4 for each group) using Qiagen RNeasy Plus Mini kit and the quality 560 assessed on a TapeStation 2200 (Agilent technologies). QuantSeq 3′ mRNA-Seq Library Prep Kit 561 FWD (Lexogen, Vienna Austria), supplemented with a common set of external RNA controls, 562 according to manufacturer’s recommendations (ERCC RNA Spike-In mix, ThermoFisher 563 Scientific, #4456740). An in-house pipeline was used for read mapping and alignment, transcript 564 construction and quantification of data generated by sequencing (HiSeq 2000, NYGC, NY, USA). 565 This procedure was done in the Epigenetics Core from MSKCC. RNA-seq data has been 566 deposited to GSE114320 and can be viewed for reviewers only: 567 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114320. 568 569 Synthesis of Ro-OH by reduction of Ro 08-2750 aldehyde 570 To a cooled (0 °C) slurry of Ro 08-2750 (19 mg, 0.070 mmol) in anhydrous MeOH (1.9 mL) was

571 added LiBH4 (32 mg, 1.5 mmol) in portions over 5 min. The slurry turned from bright orange to 572 dark brown, then dark green within 10 min. The reaction mixture was removed from the ice bath 573 and allowed to warm to rt (22 °C) over 2 h. Reaction progress was monitored by LC-MS (5–95%

574 MeCN in H2O). Four portions of LiBH4 (10 mg, 0.04 mmol) were added every 12 h until the 575 reaction was complete. The reaction was quenched with AcOH (10 mL) and filtered. The solids

576 were washed with water (5 mL), MeOH (5 mL), and Et2O (5 mL). The solid was collected and 577 dried under vacuum to provide a pale orange solid (7 mg, 26%). Purification by HPLC (5–95%

578 MeCN in H2O) afforded the product as an orange solid (3 mg, 16%). The synthesis was adapted 579 from Salach et al.6

18 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

580 1H-NMR (600 MHz, DMSO) δ 11.34 (s, 1H), 7.91 (s, 1H), 7.87 (s, 1H), 5.69 (t, J = 4.5, 1H), 4.74 581 (d, J = 4.4, 2 H), 3.99 (s, 3H), 2.38 (s, 3H). 13C-NMR (150 MHz, DMSO) 159.8 (C), 155.4 (C), 582 150.5 (C), 149.7 (C), 137.4 (C), 133.57 (C), 133.56 (C), 131.5 (CH), 131.0 (C), 112.3 (CH), 60.8

583 (CH2), 31.7 (CH3), 17.2 (CH3); IR (ATR): 2361, 2341, 1717. ESI-MS m/z (rel int): (pos) 273.1 584 ([M+H]+, 100). 585 586 Statistical analysis 587 Student's t test was used for significance testing in the bar graphs, except where stated 588 otherwise. A two-sample equal-variance model assuming normal distribution was used. The 589 investigators were not blinded to the sample groups for all experiments. P values less than 0.05 590 were considered to be significant. Graphs and error bars reflect means + standard error of the 591 mean except stated otherwise. All statistical analyses were carried out using GraphPad Prism 7.0 592 and the R statistical environment. 593

594 Modeling and System preparation for Computational Modeling

595 System preparation, modeling, and initial docking calculations were performed using the 596 Schrödinger Suite molecular modeling package (version 2015-4), using default parameters 597 unless otherwise noted. The MSI2 RRM1 protein structure (PDB ID: 6DBP) was prepared using 7 598 the Protein Preparation Wizard . In this step, force field atom types and bond orders were 599 assigned, missing atoms were added, tautomer/ionization states were assigned, water 600 orientations were sampled, and ionizable residues (Asn, Gln, and His residues) have their 601 tautomers adjusted to optimize the hydrogen bond network. A constrained energy minimization 602 was then performed. All crystallographiclly resolved water molecules were retained. 603 Potential binding sites were explored and characterized using the SiteMap8,9 tool. Ligands with 604 experimental activity and known inactives were docked into putative binding sites using Glide 605 SP10,11 to evaluate enrichment of known actives. Best docking scores were for the ‘Ro’ series for 606 the ‘(-)-gossypol’ binding site described by Lan et al.12 compared to other putative pockets. 607 Since the receptor may not be in an optimal conformation to bind small molecule inhibitors, 608 induced fit docking13 of ligand Ro 08-2750 was performed to this binding pocket. Induced fit 609 docking results were validated with the metadynamics protocol described by Clark et al.14 In 610 these metadynamics simulations a biasing potential is applied to the ligand RMSD as collective 611 variable. The resulting potential energy surface is evaluated towards how easy a ligand can move 612 away from the initial binding mode. The underlying assumption is that a ligand pose which is 613 closer to the real one has a higher energetic barrier to leave the pose than an incorrect pose. The 614 pose ranked second using the induced fit docking score retrieved the best score from the 615 metadynamics ranking protocol compared to the other induced fit docking poses. This receptor 616 configuration was furthermore tested towards its suitability for a virtual screening by a Glide SP

19 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

617 docking of known actives into this pocket. The docking scores using this receptor conformations 618 were better (down to -6.2) compared to the initial protein conformation in the crystal structure. 619 Furthermore, a WaterMap15,16 calculation was done for this receptor. 620 621 Induced Fit Docking of Ro-NGF and Ro-OH compounds 622 Induced Fit Docking (IFD) was performed against the receptor pose from the selected Ro 08- 623 2750 pose, using Schödinger molecular modeling suite (version 2017-4). Poses for Ro-NGF and 624 Ro-OH, the top and second scored poses respectively, were selected to most closely match the 625 Ro 08-2750 pose. 626 627 Alchemical Free Energy Calculations 628 Absolute alchemical free energy calculations were carried out to validate the putative binding 629 poses in a fully-flexible explicitly solvated system. The YANK GPU-accelerated free energy 630 calculation code with the Amber family of forcefields was used for this purpose. Details follow: 631 System preparation and modeling. The top poses generated by induced fit docking, as described 632 above, were selected as input protein and ligand poses. Because proteins and ligands were 633 already prepared, they were simply run through the pdbfixer 1.4 command line tool with add- 634 atoms and add-residues set to None to convert residue and atom names to be compatible with 635 Amber tleap. 636 Parameterization. tleap (from the minimal conda-installable AmberTools 16 suite ambermini 637 16.16.0) was used to solvate the complex in a cubic box with a 12Å buffer of TIP3P water 638 molecules around the protein17. The system was parameterized using AMBER’s forcefield 639 ff14sb18 and GAFF 1.819. Missing ligand parameters were determined using antechamber20. The 640 ligand was assigned charges using the AM1-BCC21,22 implementation in OpenEye (OEtoolkit 641 2017.6.123 through openmoltools 0.8.1). 642 Minimization. Minimization was performed using the implementation of the L-BFGS24 algorithm in 643 OpenMM 7.1.125 with a tolerance of 1kJ/mol/nm. 644 Production Simulation. Production simulation was run using YANK 0.19.426 using OpenMMTools 645 0.13.4. In order to keep the ligand from diffusing away from the protein while in a weakly coupled 646 state, it was confined to the binding site using a Harmonic restraint with an automatically- 647 determined force constant (K = 0.33 kcal/mol/Å2). The restraint was centered on the following 648 receptor residues using all-atom selection: 2, 4, 46, 76, 78, and 80. The ligand atoms were 649 automatically determined. The calculation was performed using particle mesh Ewald (PME)27 650 electrostatics with default YANK settings with a real-space cutoff of 9Å. A long-range isotropic 651 dispersion correction was applied to correct for truncation of the Lennard-Jones potential at 9Å. 652 The system was automatically solvated with TIP3P28 solvent and four neutralizing Cl- ions, 653 paramterized using the Joung and Cheaham paramters29. Production alchemical Hamiltonian

20 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

654 exchange free energy calculations were carried out at 300 K and 1 atm using a Langevin 655 integrator (VRORV splitting)30 with a 2 fs timestep, 5.0 ps-1 collision rate, and a molecular-scaling 656 Monte Carlo barostat. Ro 08-2750 and Ro-NGF were run for 10000 iterations (50 ns/replica) with 657 2500 timesteps (5 ps) per iteration, while Ro-OH was run for 15000 iterations (75 ns/replica) with 658 2500 timesteps (5 ps) per iteration. Complex configurations were stored for each replica once per 659 iteration. Replica exchange steps were performed each iteration to mix replicas using the Gibbs 660 sampling scheme described previously31. The alchemical pathway was automatically determined 661 for each compound using the YANK autoprotocol protocol trailblazing feature. 662 Absolute binding free energy estimates. Absolute free energies (ΔG) of binding for each 663 compound was estimated using MBAR32. Samples were reweighted to a cutoff of 16Å to correct 664 the isotropic dispersion correction to a nonisotropic long-range dispersion. This correction is 665 important to account for the heterogeneous density of protein. To remove the harmonic restraint 666 bias, samples were reweighted to substitute a squared well restraint of radius 10Å. 667 Clustering analysis. The fully interacting trajectory from YANK was extracted to a PDB file, 668 discarding the following number of initial iterations, which came prior to equilibration33: 1500 for 669 Ro 08-2750, 1600 for Ro-OH, and 1600 for Ro-NGF. These trajectories were aligned in MDTraj34 670 using only protein backbone atoms. The small molecules were then sliced out and clustered on 671 Cartesian coordinates using the MSMBuilder35 implementation of RegularSpatial clustering using 672 a 1Å RMSD cutoff. For the most populated clusters for Ro 08-2750 and Ro-OH, cluster centers 673 were selected and shown with 10 randomly sampled cluster members. Ro-NGF produced a large 674 number of lowly populated clusters with highly heterogeneous binding poses, and were therefore 675 not shown. 676 Conformational heterogeneity analysis. To investigate the conformational heterogeneity in the 677 presence or absence of the ligand, the fully interacting thermodynamic state (corresponding to 678 the holo protein bound to the ligand) and fully non-interacting state (corresponding to the apo 679 protein free of ligand interactions) for all three ligands were extracted using a 4-frame skip, 680 discarding the initial frames as above. 681 682 Code availability. All Schrödinger project files, YANK simulation inputs, and analysis scripts have 683 been made publicly available (https://github.com/choderalab/musashi). 684 685 Methods. Supplemental References. 686 1 Park, S. M. et al. Musashi2 sustains the mixed-lineage leukemia-driven stem cell 687 regulatory program. J Clin Invest 125, 1286-1298 (2015). 688 2 Minuesa, G. et al. A 1536-well fluorescence polarization assay to screen for modulators 689 of the MUSASHI family of RNA-binding proteins. Comb Chem High Throughput Screen 690 17, 596-609 (2014). 691 3 Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation 692 mode. Methods Enzymol 276, 307-326 (1997).

21 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

693 4 Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular 694 structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010). 695 5 Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building 696 protein models in electron density maps and the location of errors in these models. Acta 697 Crystallogr A 47 ( Pt 2), 110-119 (1991). 698 6 Salach, J. et al. Studies on succinate dehydrogenase. Site of attachment of the 699 covalently-bound flavin to the peptide chain. Eur J Biochem 26, 267-278 (1972). 700 7 Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and 701 ligand preparation: parameters, protocols, and influence on virtual screening 702 enrichments. J Comput Aided Mol Des 27, 221-234 (2013). 703 8 Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J 704 Chem Inf Model 49, 377-389 (2009). 705 9 Halgren, T. New method for fast and accurate binding-site identification and analysis. 706 Chem Biol Drug Des 69, 146-148 (2007). 707 10 Halgren, T. A. et al. Glide: a new approach for rapid, accurate docking and scoring. 2. 708 Enrichment factors in database screening. J Med Chem 47, 1750-1759 (2004). 709 11 Friesner, R. A. et al. Glide: a new approach for rapid, accurate docking and scoring. 1. 710 Method and assessment of docking accuracy. J Med Chem 47, 1739-1749 (2004). 711 12 Lan, L. et al. Natural product (-)-gossypol inhibits colon cancer cell growth by targeting 712 RNA-binding protein Musashi-1. Mol Oncol 9, 1406-1420 (2015). 713 13 Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A. & Farid, R. Novel procedure for 714 modeling ligand/receptor induced fit effects. J Med Chem 49, 534-553 (2006). 715 14 Clark, A. J. et al. Prediction of Protein-Ligand Binding Poses via a Combination of 716 Induced Fit Docking and Metadynamics Simulations. J Chem Theory Comput 12, 2990- 717 2998 (2016). 718 15 Young, T., Abel, R., Kim, B., Berne, B. J. & Friesner, R. A. Motifs for molecular 719 recognition exploiting hydrophobic enclosure in protein-ligand binding. Proc Natl Acad Sci 720 U S A 104, 808-813 (2007). 721 16 Abel, R., Young, T., Farid, R., Berne, B. J. & Friesner, R. A. Role of the active-site 722 solvent in the thermodynamics of factor Xa ligand binding. J Am Chem Soc 130, 2817- 723 2831, (2008). 724 17 Case, D. A., Betz, R.M., Cerutti, D.S., Cheatham, III, T.E., Darden, T.A., Duke, R.E., 725 Giese, T.J., Gohlke, H., Goetz, A.W., Homeyer, N., Izadi, S., Janowski, P., Kaus, J., 726 Kovalenko, A., Lee, T.S., LeGrand, S., Li, P., Lin, C., Luchko, T., Luo, R., Madej, R., 727 Mermelstein, D., Merz, K.M., Monard, G., Nguyen, H., Nguyen, H.T., Omelyan, I., 728 Onufriev, A., Roe, D.R., Roitberg, A., Sagui, C., Simmerling, C.L., Botello-Smith, W.M., 729 Swails, J., Walker, R.C., Wang, J., Wolf, R.M., Wu, X., Xiao, L. and Kollman, P.A. 730 AMBER 2016, University of California, San Francisco (2016). 731 18 Maier, J. A. et al. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone 732 Parameters from ff99SB. J Chem Theory Comput 11, 3696-3713 (2015). 733 19 Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and 734 testing of a general amber force field. J Comput Chem 25, 1157-1174 (2004). 735 20 Wang, J., Wang, W., Kollman, P. A. & Case, D. A. Automatic atom type and bond type 736 perception in molecular mechanical calculations. J Mol Graph Model 25, 247-260 (2006). 737 21 Jakalian, A., Jack, D. B. & Bayly, C. I. Fast, efficient generation of high-quality atomic 738 charges. AM1-BCC model: II. Parameterization and validation. J Comput Chem 23, 1623- 739 1641 (2002). 740 22 Jakalian, A., Bush, B. L., Jack, D. B. & Bayly, C. I. Fast, efficient generation of high- 741 quality atomic carges AM1-BCC model: I. Method. J Comput Chem 21, 132-146 (2000). 742 23 Toolkits, O. Oct.1 OpenEye Scientific Software, Santa Fe, NM. 743 http://www.eyesopen.com/. (2017). 744 24 Nocedal, J. American Mathematical Society. Math. Comp. 35, 773-782 (1980). 745 25 Eastman, P. et al. OpenMM 7: Rapid development of high performance algorithms for 746 molecular dynamics. PLoS Comput Biol 13, e1005659 (2017).

22 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

747 26 Wang, K., Chodera, J. D., Yang, Y. & Shirts, M. R. Identifying ligand binding sites and 748 poses using GPU-accelerated Hamiltonian replica exchange molecular dynamics. J 749 Comput Aided Mol Des 27, 989-1007 (2013). 750 27 Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N log(N) method for Ewald 751 sums in large systems J Chem Phys 98, 10089-10092 (1998). 752 28 Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. 753 Comparison of simple potential functions for simulating liquid water. J Chem Phys 79, 754 926-935 (1998). 755 29 Joung, I. S. & Cheatham, T. E., 3rd. Determination of alkali and halide monovalent ion 756 parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B 112, 757 9020-9041 (2008). 758 30 Leimkuhler, B. & Matthews, C. Efficient molecular dynamics using geodesic integration 759 and solvent-solute splitting. Proc Math Phys Eng Sci 472, 20160138 (2016). 760 31 Chodera, J. D. & Shirts, M. R. Replica exchange and expanded ensemble simulations as 761 Gibbs sampling: simple improvements for enhanced mixing. J Chem Phys 135, 194110 762 (2011). 763 32 Shirts, M. R. & Chodera, J. D. Statistically optimal analysis of samples from multiple 764 equilibrium states. J Chem Phys 129, 124105 (2008). 765 33 Chodera, J. D. A Simple Method for Automated Equilibration Detection in Molecular 766 Simulations. J Chem Theory Comput 12, 1799-1805 (2016). 767 34 McGibbon, R. T. et al. MDTraj: A Modern Open Library for the Analysis of Molecular 768 Dynamics Trajectories. Biophys J 109, 1528-1532 (2015). 769 35 Beauchamp, K. A. et al. MSMBuilder2: Modeling Conformational Dynamics at the 770 Picosecond to Millisecond Scale. J Chem Theory Comput 7, 3412-3419 (2011). 771 772

23 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

773 References 774 1 Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding 775 proteins. Nat Rev Mol Cell Biol, doi:10.1038/nrm.2017.130 (2018). 776 2 Kharas, M. G. & Lengner, C. J. Stem Cells, Cancer, and MUSASHI in Blood and Guts. 777 Trends Cancer 3, 347-356, doi:10.1016/j.trecan.2017.03.007 (2017). 778 3 Pereira, B., Billaud, M. & Almeida, R. RNA-Binding Proteins in Cancer: Old Players and 779 New Actors. Trends Cancer 3, 506-528, doi:10.1016/j.trecan.2017.05.003 (2017). 780 4 Vu, L. P. et al. Functional screen of MSI2 interactors identifies an essential role for 781 SYNCRIP in myeloid leukemia stem cells. Nat Genet 49, 866-875, doi:10.1038/ng.3854 782 (2017). 783 5 Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via 784 recruitment to DCAF15. Science 356, doi:10.1126/science.aal3755 (2017). 785 6 Ghosh, M. et al. Essential role of the RNA-binding protein HuR in progenitor cell survival 786 in mice. J Clin Invest 119, 3530-3543, doi:10.1172/JCI38263 (2009). 787 7 Palanichamy, J. K. et al. RNA-binding protein IGF2BP3 targeting of oncogenic transcripts 788 promotes hematopoietic progenitor proliferation. J Clin Invest 126, 1495-1511, 789 doi:10.1172/JCI80046 (2016). 790 8 Park, S. M. et al. Musashi2 sustains the mixed-lineage leukemia-driven stem cell 791 regulatory program. J Clin Invest 125, 1286-1298, doi:10.1172/JCI78440 (2015). 792 9 Lee, S. C. & Abdel-Wahab, O. Therapeutic targeting of splicing in cancer. Nat Med 22, 793 976-986, doi:10.1038/nm.4165 (2016). 794 10 Kanemura, Y. et al. Musashi1, an evolutionarily conserved neural RNA-binding protein, is 795 a versatile marker of human glioma cells in determining their cellular origin, malignancy, 796 and proliferative activity. Differentiation 68, 141-152 (2001). 797 11 Hemmati, H. D. et al. Cancerous stem cells can arise from pediatric brain tumors. Proc 798 Natl Acad Sci U S A 100, 15178-15183, doi:10.1073/pnas.2036535100 (2003). 799 12 Shu, H. J. et al. Expression of the Musashi1 gene encoding the RNA-binding protein in 800 human hepatoma cell lines. Biochem Biophys Res Commun 293, 150-154, 801 doi:10.1016/S0006-291X(02)00175-4 (2002). 802 13 Li, N. et al. The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal 803 Oncoproteins. Cell Rep 13, 2440-2455, doi:10.1016/j.celrep.2015.11.022 (2015). 804 14 Wang, S. et al. Transformation of the intestinal epithelium by the MSI2 RNA-binding 805 protein. Nat Commun 6, 6517, doi:10.1038/ncomms7517 (2015). 806 15 Oskarsson, T. et al. Breast cancer cells produce tenascin C as a metastatic niche 807 component to colonize the lungs. Nat Med 17, 867-874, doi:10.1038/nm.2379 (2011). 808 16 Kang, M. H. et al. Musashi RNA-binding protein 2 regulates estrogen receptor 1 function 809 in breast cancer. Oncogene 36, 1745-1752, doi:10.1038/onc.2016.327 (2017). 810 17 Wang, X. Y. et al. Musashi1 as a potential therapeutic target and diagnostic marker for 811 lung cancer. Oncotarget 4, 739-750, doi:10.18632/oncotarget.1034 (2013). 812 18 Vo, D. T. et al. The oncogenic RNA-binding protein Musashi1 is regulated by HuR via 813 mRNA translation and stability in glioblastoma cells. Mol Cancer Res 10, 143-155, 814 doi:10.1158/1541-7786.MCR-11-0208 (2012). 815 19 Guo, K. et al. The Novel KLF4/MSI2 Signaling Pathway Regulates Growth and 816 Metastasis of Pancreatic Cancer. Clin Cancer Res 23, 687-696, doi:10.1158/1078- 817 0432.CCR-16-1064 (2017). 818 20 Fox, R. G. et al. Image-based detection and targeting of therapy resistance in pancreatic 819 adenocarcinoma. Nature 534, 407-411, doi:10.1038/nature17988 (2016). 820 21 Barbouti, A. et al. A novel gene, MSI2, encoding a putative RNA-binding protein is 821 recurrently rearranged at disease progression of chronic myeloid leukemia and forms a 822 fusion gene with HOXA9 as a result of the cryptic t(7;17)(p15;q23). Cancer Res 63, 1202- 823 1206 (2003). 824 22 De Weer, A. et al. EVI1 overexpression in t(3;17) positive myeloid malignancies results 825 from juxtaposition of EVI1 to the MSI2 locus at 17q22. Haematologica 93, 1903-1907, 826 doi:10.3324/haematol.13192 (2008).

24 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

827 23 Saleki, R. et al. A novel TTC40-MSI2 fusion in de novo acute myeloid leukemia with an 828 unbalanced 10;17 translocation. Leuk Lymphoma 56, 1137-1139, 829 doi:10.3109/10428194.2014.947611 (2015). 830 24 Wang, K. et al. Patient-derived xenotransplants can recapitulate the genetic driver 831 landscape of acute leukemias. Leukemia 31, 151-158, doi:10.1038/leu.2016.166 (2017). 832 25 Ito, T. et al. Regulation of myeloid leukaemia by the cell-fate determinant Musashi. 833 Nature 466, 765-768, doi:10.1038/nature09171 (2010). 834 26 Kharas, M. G. et al. Musashi-2 regulates normal hematopoiesis and promotes aggressive 835 myeloid leukemia. Nat Med 16, 903-908, doi:10.1038/nm.2187 (2010). 836 27 Thol, F. et al. Prognostic significance of expression levels of stem cell regulators MSI2 837 and NUMB in acute myeloid leukemia. Ann Hematol 92, 315-323, doi:10.1007/s00277- 838 012-1637-5 (2013). 839 28 Taggart, J. et al. MSI2 is required for maintaining activated myelodysplastic syndrome 840 stem cells. Nat Commun 7, 10739, doi:10.1038/ncomms10739 (2016). 841 29 Byers, R. J., Currie, T., Tholouli, E., Rodig, S. J. & Kutok, J. L. MSI2 protein expression 842 predicts unfavorable outcome in acute myeloid leukemia. Blood 118, 2857-2867, 843 doi:10.1182/blood-2011-04-346767 (2011). 844 30 Kwon, H. Y. et al. Tetraspanin 3 Is Required for the Development and Propagation of 845 Acute Myelogenous Leukemia. Cell Stem Cell 17, 152-164, 846 doi:10.1016/j.stem.2015.06.006 (2015). 847 31 Park, S. M. et al. Musashi-2 controls cell fate, lineage bias, and TGF-beta signaling in 848 HSCs. J Exp Med 211, 71-87, doi:10.1084/jem.20130736 (2014). 849 32 Rentas, S. et al. Musashi-2 attenuates AHR signalling to expand human haematopoietic 850 stem cells. Nature 532, 508-511, doi:10.1038/nature17665 (2016). 851 33 Kudinov, A. E., Karanicolas, J., Golemis, E. A. & Boumber, Y. Musashi RNA-Binding 852 Proteins as Cancer Drivers and Novel Therapeutic Targets. Clin Cancer Res 23, 2143- 853 2153, doi:10.1158/1078-0432.CCR-16-2728 (2017). 854 34 Sakakibara, S., Nakamura, Y., Satoh, H. & Okano, H. Rna-binding protein Musashi2: 855 developmentally regulated expression in neural precursor cells and subpopulations of 856 neurons in mammalian CNS. J Neurosci 21, 8091-8107 (2001). 857 35 Zearfoss, N. R. et al. A conserved three-nucleotide core motif defines Musashi RNA 858 binding specificity. J Biol Chem 289, 35530-35541, doi:10.1074/jbc.M114.597112 (2014). 859 36 Ohyama, T. et al. Structure of Musashi1 in a complex with target RNA: the role of 860 aromatic stacking interactions. Nucleic Acids Res 40, 3218-3231, 861 doi:10.1093/nar/gkr1139 (2012). 862 37 Katz, Y. et al. Musashi proteins are post-transcriptional regulators of the epithelial-luminal 863 cell state. Elife 3, e03915, doi:10.7554/eLife.03915 (2014). 864 38 Minuesa, G. et al. A 1536-well fluorescence polarization assay to screen for modulators 865 of the MUSASHI family of RNA-binding proteins. Comb Chem High Throughput Screen 866 17, 596-609 (2014). 867 39 Eibl, J. K., Strasser, B. C. & Ross, G. M. Identification of novel 868 pyrazoloquinazolinecarboxilate analogues to inhibit nerve growth factor in vitro. Eur J 869 Pharmacol 708, 30-37, doi:10.1016/j.ejphar.2013.03.029 (2013). 870 40 Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for 871 interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545- 872 15550, doi:10.1073/pnas.0506580102 (2005). 873 41 Zhang, H. et al. Musashi2 modulates K562 leukemic cell proliferation and apoptosis 874 involving the MAPK pathway. Exp Cell Res 320, 119-127, 875 doi:10.1016/j.yexcr.2013.09.009 (2014). 876 42 Han, Y. et al. Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia 877 and Enhances Chemosensitivity to Daunorubicin. PLoS One 10, e0136484, 878 doi:10.1371/journal.pone.0136484 (2015). 879 43 Meisner, N. C. et al. Identification and mechanistic characterization of low-molecular- 880 weight inhibitors for HuR. Nat Chem Biol 3, 508-515, doi:10.1038/nchembio.2007.14 881 (2007).

25 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

882 44 Wu, X. et al. Identification and validation of novel small molecule disruptors of HuR- 883 mRNA interaction. ACS Chem Biol 10, 1476-1484, doi:10.1021/cb500851u (2015). 884 45 Lan, L. et al. Natural product (-)-gossypol inhibits colon cancer cell growth by targeting 885 RNA-binding protein Musashi-1. Mol Oncol 9, 1406-1420, 886 doi:10.1016/j.molonc.2015.03.014 (2015). 887 46 Lim, D., Byun, W. G., Koo, J. Y., Park, H. & Park, S. B. Discovery of a Small-Molecule 888 Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically 889 Labeled Lin28. J Am Chem Soc, doi:10.1021/jacs.6b06965 (2016). 890 47 Roos, M. et al. A Small-Molecule Inhibitor of Lin28. ACS Chem Biol 11, 2773-2781, 891 doi:10.1021/acschembio.6b00232 (2016). 892 48 Jarvis, W. D., Turner, A. J., Povirk, L. F., Traylor, R. S. & Grant, S. Induction of apoptotic 893 DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by 894 pharmacological inhibitors of protein kinase C. Cancer Res 54, 1707-1714 (1994). 895 49 Zhu, J. et al. Niemann-Pick C2 Proteins: A New Function for an Old Family. Front Physiol 896 9, 52, doi:10.3389/fphys.2018.00052 (2018). 897 50 Judge, J. L. et al. The Lactate Dehydrogenase Inhibitor Gossypol Inhibits Radiation- 898 Induced Pulmonary Fibrosis. Radiat Res 188, 35-43, doi:10.1667/RR14620.1 (2017). 899 51 Zeng, Y., Ma, J., Xu, L. & Wu, D. Natural Product Gossypol and Its Derivatives in 900 Precision Cancer Medicine. Curr Med Chem, doi:10.2174/0929867324666170523123655 901 (2017). 902 52 Clingman, C. C. et al. Allosteric inhibition of a stem cell RNA-binding protein by an 903 intermediary metabolite. Elife 3, doi:10.7554/eLife.02848 (2014). 904 53 Sadlish, H. et al. Evidence for a functionally relevant rocaglamide binding site on the 905 eIF4A-RNA complex. ACS Chem Biol 8, 1519-1527, doi:10.1021/cb400158t (2013). 906 54 Choo, A. Y., Yoon, S. O., Kim, S. G., Roux, P. P. & Blenis, J. Rapamycin differentially 907 inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. 908 Proc Natl Acad Sci U S A 105, 17414-17419, doi:10.1073/pnas.0809136105 (2008). 909 55 Fang, T. et al. Musashi 2 contributes to the stemness and chemoresistance of liver 910 cancer stem cells via LIN28A activation. Cancer Lett 384, 50-59, 911 doi:10.1016/j.canlet.2016.10.007 (2017). 912 56 Sheng, W. et al. Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance 913 and malignant biology of pancreatic cancer. FASEB J 31, 2429-2438, 914 doi:10.1096/fj.201601240R (2017). 915

26 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

916 Figure Legends

917 Figure 1. Ro 08-2750 (Ro) is a novel selective MSI RNA-binding activity inhibitor.

918 (a) Fluorescence polarization secondary validation of Ro 08-2750 (Ro) IC50 for MSI-RNA binding 919 inhibition in 384-well format. Seven independent experiments performed in duplicate ± standard 920 error mean (s.e.m.) are shown; (b) Representative Electrophoresis Mobility Shift Assays (EMSA) 921 for GST- and GST-MSI2 proteins (125 and 250ng) using biotinylated-RNA oligo in the absence or 922 presence of unlabeled RNA (left); quantification of MSi2-RNA complexes of at five independent 923 experiments ± s.e.m. is shown in bar graph (right); (c) EMSA for GST-MSI2 (125ng) in the 924 presence of increasing concentrations of Ro (5 to 40 μM); quantification of RNA-protein 925 complexes of at least four independent experiments ± s.e.m. is shown in bar graph (right); (d) 926 Microscale Thermophoresis (MST) assay showing interaction of Ro with GST-MSI2, GST- 927 MSI2/RNA complexes or the RRM-RBP control GST-SYNCRIP. Ro concentrations ranged from

928 0.0153 to 500 μM. Affinity (KD) values ± s.e.m. (μM) of three independent experiments are shown 929 as percentage of fraction bound. For (b) and (c): two-tailed Paired t-test; *p<0.05; **p<0.01, 930 ***p<0.005, ****p<0.001.

931

932 Figure 2. Ro 08-2750 interacts with the RNA-recognition motif 1 (RRM1) of MSI2 and its 933 analogs show minimal or null residual activity.

934 (a) Global front view of the docked Ro 08-2750 (Ro) molecule in the RNA-binding site of human 935 MSI2 RRM1 based on the X-ray diffraction crystal structure obtained at 1.7Å resolution (RCSB 936 PDB 6DBP); (b) Lateral and close up (inset) view of Ro showing the most relevant interaction 937 residues (F66, F97 and R100) and the distances (Å) between them and Ro closest atoms; (c) 2D 938 representation of residues involved in Ro binding showing F66 (hydrophobic stacking), K22 (H- 939 bonding), F97 (H-bonding with the backbone) and R100 (π-cation interaction) from RRM1 as 940 main interaction partners; (d) Microscale Thermophoresis (MST) assay showing affinity of 941 interaction of Ro with full-length GST-MSI2 WT (red), GST-MSI2 F97A (cyan) and GST-

942 F66A/F97A/R100A (orange). KD values ± standard deviation (μM) of at least three independent 943 experiments are shown as percentage of fraction bound; (e) Chemical structures of Ro analogues

944 used in (f), (g), (h) and (i) panels. Ro-NGF (high affinity Neural Growth Factor -NGF- inhibitor, KD -6 945 (NGF) = 1.7x10 M) and Ro-OH (reduced form of Ro); (f) The cluster centers for Ro (left), RoOH, 946 (center) and Ro-NGF (right), derived using regular spatial clustering with a ligand RMSD cutoff of 947 1Å. Ro-NGF (right) showing a much larger number of clusters than Ro 08-2750 (left) or RoOH 948 (center). (g) Representative EMSA for GST-MSI2 (125ng) in the absence (DMSO) or presence of 949 Ro (20 μM), Ro-OH (20 μM) or unlabeled RNA oligo (1 μM) and quantification of RNA-protein 950 complexes of at least three independent experiments (bar graph, below); (h) Representative

27 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

951 EMSA for GST-MSI2 (125ng) in the absence (DMSO) or presence of Ro (20 μM), Ro-NGF (20 952 μM) or unlabeled RNA oligo (1 μM) and quantification of RNA-protein complexes of at least three 953 independent experiments (bar graph, below) ± s.e.m.; (i) MST assays showing interaction of Ro,

954 Ro-OH and Ro-NGF with GST-MSI2 WT. Drug concentrations ranged from 0.0153 to 500 μM. KD 955 values ± standard deviation (μM) of at least three experiments are shown as percentage of 956 fraction bound; For (g) and (h), two-tailed Paired t-test; ns, not significant, *p<0.05, **p<0.01, 957 ***p<0.005.

958 959 Figure 3. Ro 08-2750 treatment leads to preferential increase in differentiation and 960 apoptosis in murine MLL-AF9 leukemic cells compared to Lin-Sca+cKit+ (LSK) cells. 961 (a) Cytotoxicity assay (Cell-Titer Glo®) of Ro (red), Ro-OH (cyan) and Ro-NGF (orange) in MLL-

962 AF9+ BM cells. 50% Effective Concentration (EC50) values, average of at least three independent 963 experiments ± standard deviation are shown. (b) Flow cytometry representative histograms of 964 DMSO (grey) and 5 μM Ro (red) treated MLL-AF9+ BM cells showing myeloid differentiation 965 markers (Mac1 and Gr1); bar graphs (below) show average (fold change increase) ± standard 966 error mean of three independent experiments, performed in triplicate. Paired t-test, *p<0.05; 967 **p<0.01. (c) Representative immunocytochemistry images of cytospun MLL-AF9+ BM cells 968 control (DMSO) or Ro treated (5 and 10 μM) and stained by Eosin Y and Methylene Blue/ Azure 969 A. Scale, 50 μm. (d) Apoptosis analysis by Annexin V+ (% population) for MLL-AF9+ BM cells 970 cultured in absence (DMSO, black) or presence of Ro 5 μM (light red) or 10 μM (red). Results 971 represent at least three independent experiments ± s.e.m.. (e) Colony Formation Unit (CFU) 972 assay of MLL-AF9+ BM cells transduced with MSCV-IRES-BFP (MIB, control) or MSCV-IRES- 973 MSI2-BFP (MSI2-BFP) retroviral vectors. Results represent the average ± s.e.m. of colony 974 numbers of at least five experiments performed in duplicate. (f) Representative immunoblot of 975 MLL-AF9+ BM MIB (black bars) and MSI2-BFP (red bars) cells (used in panel e) after DMSO or 976 10 μM Ro treatment for 4h. β-ACTIN, loading control. (g) CFU assay of Lin-Sca+cKit+ (LSK) 977 versus MLL-AF9+ BM cells demonstrates Ro 08-2750 therapeutic window. Results represent the 978 average ± s.e.m. of colony numbers of three experiments performed in duplicate. Two tailed 979 Paired t-test (b, d, e and g), *p<0.05, **p<0.01, ***p<0.005.

980

981 Figure 4. Ro 08-2750 treatment inhibits survival of human AML cell lines and patient cells.

982 (a) Cytotoxicity assay (Cell-Titer Glo®) of Ro, Ro-OH and Ro-NGF in MOLM13 and K562 cells.

983 EC50 values average of three independent experiments ± standard deviation is shown. (b) Mean 984 Fluorescence Intensity (MFI) fold changes of CD14 (myeloid marker, MOLM13) and CD235a 985 (Glycophorin-A; erythroid marker, K562) after 48h treatment with DMSO (control, black bars) or

28 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

986 Ro 20 μM (red bars). Data is normalized to DMSO control cells. Representative histograms are 987 shown in Extended Data Figure 6a. (c) Representative immunocytochemistry images of cytospun 988 MOLM13 and K562 cells treated for 48h with DMSO (control) or Ro 20 μM and stained with Eosin 989 Y and Methylene Blue/ Azure A. Scale, 20 μm. (d) Apoptosis analysis by Annexin V+ (% 990 population). MOLM13 and K562 were cultured in DMSO (black bars) or in the presence of Ro 20 991 μM (red bars) for the indicated times and Annexin V positivity and 7AAD was measured. Results 992 represent three independent experiments ± standard deviation. (e) CFU assay of MOLM13 and 993 K562 in the presence of Ro 08-2750 at different concentrations (1, 5, 10 and 20 μM). Data is 994 shown as average colony numbers (normalized to DMSO control) ± s.e.m. of at least three 995 independent experiments. (f) CFU assay of cord-blood derived CD34+ HSPCs and AML patient 996 BM cells. Data is shown as average colony numbers (normalized to DMSO) ± s.e.m. of three 997 different blood donors for CD34+ and three independent AML patients. Two tailed Paired t-test 998 (DMSO vs Ro treated, unless indicated with lines); *p<0.05; **p<0.01; ***p<0.005, ****p<0.001.

999

1000 Figure 5. Ro 08-2750 treatment resembles gene signature from MSI2 depleted cells and 1001 demonstrates inhibition of MSI2 target translation.

1002 (a) Scheme of RNA-immunoprecipitation (IP) protocol followed with K562-MIG (MSCV-IRES- 1003 GFP) or FLAG-MSI2 overexpresing cells. (b) Ro 08-2750 inhibitory effect in the RNA-IP 1004 enrichment of MSI2 mRNA targets in K562-FLAG-MSI2 versus K562-MIG after 1h treatment at 1005 10 μM. Data is shown as average of inhibition effect (normalized to DMSO cells) ± s.e.m. of four 1006 independent experiment. (c) Up-regulated and down-regulated gene sets obtained by RNA-seq 1007 analysis after 20 μM Ro 4h treatment in K562 and MOLM13 cells showing identical signature as 1008 previously obtained using shRNA against MSI2 in CML-BC and AML lines26. (d) Venn diagram 1009 showing gene Set Enrichment Analysis (GSEA) overlap between MOLM13 (red), K562 (blue) 1010 (after 20 μM Ro 4h treatment) and AML/CML-BC cell lines MSI2 depleted with shRNAs (yellow) 1011 from26. Bold values inside brackets below each grup are total gene sets numbers. (e) 1012 Representative immunoblot for K562 treated with Ro at different concentrations (1, 5, 10 and 20 1013 μM) for 4h showing expression of MSI2 targets. HOXA9 is not expressed in this BCR-ABL+ 1014 (CML-BC) leukemia cell line. (f) Representative immunoblot for MOLM13 treated with Ro at 1015 different concentrations for 4h showing expression of MSI2 targets. (g) Representative 1016 immunoblot for K562 treated with Ro 20 μM at different time points (1, 4, 12 and 24h) showing 1017 expression of same MSI2 targets as in panel (e). P21 and β-ACTIN from a different 1018 representative gel are shown. (h) Representative immunoblot for MOLM13 treated with Ro 20 μM 1019 at different time points showing effect on MSI2 targets.

1020

29 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1021 Figure 6. Ro 08-2750 demonstrates efficacy in inhibiting leukemogenesis in short-time and 1022 long-term treatment in a MLL-AF9 in vivo model.

1023 (a) Scheme of pharmacodynamics marker experiments with Ro short-time points performed with 1024 MLL-AF9+ secondary BM cells. 10,000 MLL-AF9 GFP+ cells were transplanted and, after 3 1025 weeks, mice were injected with DMSO or Ro (13.75 mg/kg) and were sacrificed for analysis after 1026 4h and 12h (b) Surface flow analysis of c-Kit receptor in spleen cells of Ro at 4h and 12h versus 1027 DMSO treated mice. Results are represented as MFI of cKit-PE-Cy7 normalized to DMSO group. 1028 Each data point is an independent treated mouse. Mean ± s.e.m. is shown. (c) Intracellular (IC) 1029 flow analysis of c-MYC expression in spleen cells of Ro at 4h and 12h versus DMSO treated 1030 mice. Results are represented as MFI of c-MYC normalized to DMSO group. Each data point is 1031 an independent treated mouse. Mean ± s.e.m. is shown; (a-c, DMSO and Ro 4h, n=9; Ro 12h, 1032 n=6). (d) Scheme of in vivo Ro treatment in MLL-AF9+ model of myeloid leukemia. 10,000 MLL- 1033 AF9 GFP+ cells were transplanted and after 3 days, mice were injected with DMSO or Ro 13.75 1034 mg/kg (in DMSO) intraperitoneally (IP) at days 1, 4, 7, 10 and 13 (one day on, two days off drug). 1035 At day 19 of treatment, mice were sacrificed for organ weight and flow cytometry analysis of 1036 disease burden and MSI2 target, c-MYC. (e) Spleen weights at time of sacrifice. Results are 1037 represented in weight (g) and each data point represents an individual DMSO or Ro treated 1038 mouse. (f) White blood cell (WBC) counts (K/μL) at time of sacrifice. Each data point represents 1039 an individually treated mouse. (g) Intracellular (IC) flow analysis of c-MYC expression in spleen 1040 cells of Ro vs DMSO treated mice. Results are represented as % frequency (% freq) of c-MYC+ 1041 cells. Each data point is an independent treated mouse. Mean ± s.e.m. is shown. (d-g, DMSO, 1042 n=9; Ro, n=8). For all graphs, Unpaired t-test; *p<0.05, **p<0.005.

1043

30 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1044 Extended Data Figure Legends

1045 Extended Data Figure 1. Ro 08-2750 (Ro) binds to RRM1 and SYNCRIP RRM identities.

1046 (a) MicroScale Thermophoresis (MST) assay showing interaction of Ro with GST-RRM1 (hMSI2).

1047 Ro concentrations ranged from 0.0153 to 500 μM. KD values ± s.e.m. (μM) of at least three 1048 experiments are shown as percentage of fraction bound. (b) Sequence alignment of RRM1 1049 (above) and RRM2 (below) of human MSI2, MSI1 and SYNCRIP. Numbers indicate crucial RNA- 1050 binding conserved residues (in bold red) in hMSI2 (e.g. F24, corresponding to F23 in hMSI1, 1051 F165 in SYNCRIP). Grey highlights indicate conserved amino acids.

1052

1053 Extended Data Figure 2. Ro 08-2750 docking and interacting residues in comparison with 1054 Ro-OH and Ro-NGF in the RNA-binding site of RRM1.

1055 (a) Ro docked in the RNA-binding site of MSI2 RRM1 with interacting residues. Distances shown 1056 in Å; (b) MST experiments showing GST-MSI2 WT (red), F97A (cyan) and Triple

1057 (F66A/F97A/R100, orange) mutants interaction to MSI2 RNA oligo (4 MSI motifs; 15-nt). KD 1058 values ± s.e.m. of at least three experiments are shown (μM); (c) Chemical synthesis scheme of 1059 Ro-OH from Ro 08-2750 compound (see Methods); (d) FP confirmation of Ro, Ro-OH and Ro-

1060 NGF MSI2-RNA binding inhibition in 384-well format. IC50 values of two independent experiments 1061 performed in triplicate with s.e.m., 2.0±0.3 μM (Ro, red) and 25.0±8.0 μM (RoOH, cyan). Ro-NGF 1062 (orange) showed null inhibition of RNA-binding activity; (e) Docked pose of Ro-OH in the RNA 1063 binding site of MSI2 RRM1. Distances in Å; (f) Docked pose of Ro-NGF in the RNA binding site of 1064 MSI2 RRM1 showing a displaced center of the small-molecule from the binding site; (g) 2D 1065 representation of Ro-OH docked pose in the RRM1 of MSI2; (h) 2D representation of Ro-NGF 1066 docked pose in the RRM1 of MSI2 showing H-bonding of K22 changing from the O to the N in the 1067 middle ring, and π-cation interacting with R100 displaced with respect to Ro (see Figure 2b).

1068

1069 Extended Data Figure 3. 1H NMR (a) and NMR 13C spectrum (b) of Ro-OH, the synthesized 1070 reduced form of Ro.

1071

1072 Extended Data Figure 4. Alchemical free energy calculations show that both protein and 1073 ligands adopt a conformationally heterogeneous ensemble of binding poses.

1074 (a) Computed binding free energy (ΔGbind, kcal/mol) estimates from alchemical free energy 1075 calculations (y-axis) for Ro, Ro-OH, and Ro-NGF for different definitions of the “bound” complex 1076 as a function of distance cutoff (x-axis, in Å). Reported statistical errors and error bars correspond

1077 one standard error. The inset ΔGbind was calculated for a cutoff of 20Å. (b) In the alchemical

31 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1078 Hamiltonian replica exchange simulations, a conformational change is induced when MSI2 is 1079 bound (“Complex”; green) to Ro (right) or Ro-OH (center), as compared to apo MSI2 (“Apo”; 1080 gray). Ro-NGF (left) does not induce the same conformational change. (c) The top three most 1081 populous clusters for Ro 08-2750. The protein structure and solid-color ligand pose depict cluster 1082 centers, while transparent ligand poses depict 10 randomly sampled frames assigned to that 1083 cluster. Sidechains within 4Å of any of the ligands are shown as lines. (d) The top four most 1084 populous clusters for Ro-OH, using the same depiction scheme as (c).

1085

1086 Extended Data Figure 5. Flow cytometry plots showing apoptosis in MLL-AF9 leukemic 1087 cells after treatment with Ro 08-2750.

1088 Apoptosis plots (graphs in Figure 3d) showing Annexin V+ and 7AAD (live/dead staining) by 1089 Apoptosis MUSE® Cell kit and MUSE® Cell Analyzer (Millipore-Sigma) in MLL-AF9+ BM cells at 1090 8, 16, 24 and 48 hours post treatment with Ro 5 and 10 μM.

1091

1092 Extended Data Figure 6. Differentiation and apoptosis are induced in MOLM13 and K562 1093 cells after Ro 08-2750 treatment.

1094 (a) Representative histograms showing CD14 and CD13 myeloid markers in MOLM13 and 1095 erythroid differentiation markers CD235a (Glycophorin-A) and CD71 in K562 after 48h of 20 μM 1096 Ro treatment. (b) Mean Fluorescence Intensity (MFI) fold changes of CD13 (myeloid marker, 1097 MOLM13) and CD71 (erythroid marker, K562) after 48h treatment of leukemia cell lines with 1098 DMSO (control, black bars) or Ro 20 μM (red bars). Data is shown as average (normalized to 1099 DMSO control cells) ± standard error mean of three independent experiments performed in 1100 triplicate. Paired t-test (DMSO vs Ro treated); *p<0.05. (c) Apoptosis plots (from graphs in Figure 1101 4d) showing Annexin V+ and 7AAD (live/dead staining) in MOLM13 and K562 by MUSE® Cell 1102 Analyzer (Millipore-Sigma) in DMSO and Ro 20 μM treatments at 48, 72 and 96h.

1103

1104 Extended Data Figure 7. Ro effects on global translation and mRNA of MSI2 targets

1105 (a) OP-Puromycin incorporation to assess global translation rates in MOLM13 leukemia cells. 1106 Results are represented as average of Alexa Fluor 647 (AF647) Mean Fluorescence Intensity 1107 (MFI) normalized to DMSO control cells ± standard error mean of four independent experiments 1108 performed in duplicate. Paired t-test (DMSO vs Ro treated); ns, non-significant, **p<0.005. (b) 1109 Expression levels of mRNA targets of MSI2 by qPCR in K562 and (c) MOLM13. Cells were 1110 treated for 4h at 20 μM Ro. Results represent the average of ten independent experiments ± 1111 standard error mean. Paired t-test (DMSO vs Ro treated); *p<0.05.

32 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1112

1113 Extended Data Figure 8. No toxicity of Ro 08-2750 after in vivo treatment of MLL-AF9 mice.

1114 (a) Mice weight in DMSO (cyan lines, left panel) and Ro 13.75 mg/kg (orange lines, right panel) 1115 groups during the duration of the in vivo experiment. (b) Red Blood Cell (RBC) counts (M/ μL) at 1116 time of sacrifice. Each data point represents an individually treated mouse. Unpaired t-test; ns, 1117 non-significant. (c) Platelets counts (PLT) counts (K/ μL) at time of sacrifice. Each data point 1118 represents an individually treated mouse. Unpaired t-test; ns, non-significant. DMSO, n=9; Ro, 1119 n=8.

1120

33 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1121 Acknowledgements

1122 We would like to thank the current members of the M. Kharas and R. Levine labs, R. Levine, P. 1123 Lito, J. Vidigal, P. Rocha, C. Lengner, A. Kentsis and K. Keshari for their critical advice and 1124 helpful suggestions. H. Djaballah, R. Garippa and the members of the former High-Throughput 1125 Screening Core and current RNAi Core (MSKCC) for their technical and project support 1126 throughout the screen and secondary validation. We would like to thank R. Sridharan, Y. 1127 Shamay, D. Heller (MSKCC) and Rui Liang (Tri-TDI) for their scientific advice and technical 1128 support. We would also like to thank A. Gruet and the Epigenetics core for his technical support 1129 with RNA-sequencing library preparation and J. Charavalli (HTSRC, The Rockefeller University) 1130 for her technical support with initial FP measurements. M.G.K. was supported by US National 1131 Institutes of Health National Institute of Diabetes, Digestive and Kidney Diseases Career 1132 Development Award, NIDDK NIH R01-DK101989-01A1, NCI 1R01CA193842-01, Louis V. 1133 Gerstner Young Investigator Award, American Society of Hematology Junior Scholar Award, 1134 Kimmel Scholar Award, V-Scholar Award, Geoffrey Beene Award and Alex's Lemonade Stand A 1135 Award and the Starr Foundation. The research was funded by Mr. William H. Goodwin and Mrs. 1136 Alice Goodwin and the Commonwealth Foundation for Cancer Research and The Center for 1137 Experimental Therapeutics at Memorial Sloan Kettering Cancer Center. The research was funded 1138 in part through NIH/NCI Cancer Support Core Grant P30 CA08748 to M.G.K. J.D.C. 1139 acknowledges support from the Sloan Kettering Institute, a Louis V. Gerstner Young Investigator 1140 Award, NIH grant P30 CA008748, and NIH grant R01 GM121505. L.N.N. acknowledges support 1141 from Merck KGaA to support the development of open source tools for GPU-accelerated 1142 alchemical free energy calculations. M.C.P was supported by a Medical Scientist Training 1143 Program grant from the National Institute of General Medical Sciences of the National Institutes of 1144 Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri- 1145 Institutional MD-PhD Program. The MSKCC structural biology core laboratory is supported by 1146 National Cancer Institute grant P30-CA008748. X-ray diffraction data were collected at 1147 synchrotron facilities supported by grants and contracts from the National Institutes of Health 1148 (P41GM103403, HEI-S10RR029205) and the Department of Energy (DE-AC02-06CH11357).

1149

1150 Author Contributions

1151 G.M. led the project, performed experiments, analyzed data and wrote the manuscript. M.G.K. 1152 directed the project, analyzed data and wrote the manuscript. S.A., D.C., T.B., A.R., L.N., J.C. 1153 performed experiments, analyzed data and provided project support. A.C., A.S., S.M.P., T.C., J.T. 1154 performed experiments and analyzed data. M.C.P., L.F., C.L. analyzed data. J.S., C.F., M.P. 1155 provided clinical data and analysis. C.Z.R. and D.T. performed experiments and provided critical

34 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1156 reagents. J.E. and G.M.R. provided critical reagents. Y.G. performed experiments and analyzed 1157 data. C.A. and J.F.G. provided suggestions, project support and assisted analyzing data.

1158

1159 Author Information

1160 J.D.C. is a member of the Scientific Advisory Board for Schrödinger.

1161 1162 1163

35 bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1

a b

GST 125 ng 250 ng+unlabeled RNA 240 1.2 220 1.1 MSI2-RNA 200 complexes > 1.0 (mP) 180 0.9 160 0.8 140 0.7 120 O 0.6 N N O 100 H 0.5

NH Band intensity 80 N 0.4 O 0.3

60 (normalized to DMSO control) Ro 08-2750 (Ro) 40 0.2 *** IC = 2.7 ± 0.4 µM Fluorescence Polarization 20 50 0.1 Free RNA > 0 0.0 10-2 10-1 100 101 102 GST Compound (µM) 125 ng250 ng

c d +unlabeled RNA

DMSO5 µM Ro10 µM 20Ro µM40 Ro µM Ro 125 1.2 MSI2 [KD = 12.3 ± 0.5 µM] MSI2-RNA MSI2/RNA [K = 27.5 ± 2.2 µM] complexes > 1.1 D SYNCRIP [K = 236 ± 167.1 µM] 1.0 100 D 0.9 * 0.8 ** 75 0.7 0.6 *** 0.5 50 Band intensity 0.4 **** 0.3 Fraction bound (%) (normalized to DMSO control) 25 0.2 0.1 Free RNA > 0.0 0 10-2 10-1 100 101 102 103

DMSO 5 µM Ro Ro (µM) 10 µM20 Ro µM40 Ro µM Ro bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2

a b c Ro 08-2750 (Ro)

R100 F97

3.1 3.5 3.0 RRM1 (MSI2) F66

d e f

WT [KD = 10.5 ± 0.3 µM] 125 F97A [KD = 69.5 ± 14.7 µM] OH F66A/F97A/R100A [KD = no binding] N N O Ro 08Ro-2750 Ro-OHRo-OH RoRo-NGF-NGF 100 NH (%) N 75 O Ro-OH 50 H N N N O

Fraction bound 25 O N N 0 O Ro-NGF 10-2 10-1 100 101 102 103 (

Ro 08-2750 (µM) +489+ 7(

23456 !"#$%"&'()(*+(,-$'./,()(*+01()( g h i

DMSO + Ro + Ro-NGF+ unlabeled RNA DMSO + Ro + Ro-OH+ unlabeled RNA Ro [KD = 11.2 ± 0.6 µM] RNA-MSI2 RNA-MSI2 Ro-OH [K = 302.0 ± 119 µM] complex > complex > 100 D Ro-NGF [KD = no binding]

ns ** * 1.25 1.25 75 ** 1.00 1.00 50 * 0.75 0.75

Fraction bound (%) 25 0.50 0.50 *** *** Band intensity Band intensity 0.25 0.25 0 (normalized to DMSO control) 0.00 (normalized to DMSO control) 0.00 *:$;23(<=8>?(3+(( %"&'(54+3:(@+AB4:CD( 10-2 10-1 100 101 102 103 Compound (µM) d Figure 3 a Annexin V+ (%) % viability 100 100 20 40 60 80 10 20 30 40 50 60 70 80 90 0 0 10 -3 h1h2h48h 24h 16h 8h * bioRxiv preprint Ro-NGF [ Ro-OH [ Ro [ 10 µMRo 5 µMRo DMSO * 10 EC -2 50 EC = 2.6± EC 50 * 50 = 21.5± Compound ( *** = 65.2± 10 0.1µ doi: -1 certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/321174 0.8µ M] 6.5µ ** *** M] 10 M] µ 0 M) * 10 * 1 e

Colony number 10 2 (normalized) ; this versionpostedMay14,2018. 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Fold change Counts

*** (Mac1-PE MFI)

DMSO 0.0 0.5 1.0 1.5 2.0 2.5 3.0 5 µMRo DMSO Mac1-PE ** 1 µM DMSO 5 µMRo Ro 08-2750

** Ro ** MSI2-BFP 5 µM MIB

Fold change The copyrightholderforthispreprint(whichwasnot 10 µM (Gr1-APC MFI) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 f Gr1-APC MIB * MSI2 - BFP c HOXA9 cMYC SMAD3 ACTIN MSI2 g Ro 5µM Colony number (normalized) 0.00 0.25 0.50 0.75 1.00 1.25 1.50 DMSO DMSO LSK MLL-AF9+

1 µM Ro 10µM

Ro 08-2750 Ro

5 µM * ns

10 µM ** a Figure 4 d % viability Annexin V+ (%) 100 120 20 40 60 80 0 10 20 30 40 50 60 70 80 10 0 -2 8 2 96h 72h 48h Ro-NGF [ Ro-OH [ Ro [ *** bioRxiv preprint EC DMSO 20 µMRo 10 50 EC MOLM13 -1 = 8.6±1.1µM] EC Compound ( 50 50 = 35.8±1.2µM] MOLM13 = 70.1±1.0µM] ** 10 0 doi: µ certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. ** M) https://doi.org/10.1101/321174 10

Annexin V+ (%) 1 10 20 30 40 50 60 70 80 0 10 8 2 96h 72h 48h 2 * 20 µMRo DMSO % viability 100 120 20 40 60 80 K562 0 10 -2 * ; Ro-NGF [EC Ro-OH [EC Ro [ this versionpostedMay14,2018. EC 10 50 ** -1 = 8.3±1.1µM] Compound ( 50 50 = 36.0±1.1µM] = 45.7±1.0µM] e K562 Colony number (normalized) 10 0 0.00 0.25 0.50 0.75 1.00 1.25 µ M)

DMSO 10 1 MOLM13

Ro 08-2750 1 µM

* Ro

5 µM The copyrightholderforthispreprint(whichwasnot 10 * b 10 µM 2

20 µM * Fold change (MFI, normalized) Colony number (normalized) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 MOLM13 0.00 0.25 0.50 0.75 1.00 1.25 CD14

DMSO 20 µM Ro ** DMSO 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Ro 08-2750

1 µM K562

* Ro GlycA

5 µM K562 DMSO **** * 10 µM 20 µM Ro

20 µM * c f Colony number MOLM13 (normalized) K562 0.00 0.25 0.50 0.75 1.00 1.25 1.50 MORo20 DMSO DMSO AML CD34+

1 µM ** ** Ro 08-2750 *

5 µM ** Ro

10 µM * µ M 20 µM ** bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5

a b DMSO 1.4 Ro 10 uM a" 1.1. DMSO DMSO 2.2. Ro MSI-inhibitor 10 µM 1.2 * qPCR 1.0 K562 cells MSI2 Targets 1.Empty vector 1 hour Cell Lysis IP: FLAG-MSI2 Isolate RNA 0.8 2. FLAG-MSI2 0.6

0.4 Ro 08-2750 inhibition 0.2 (normalized to K562-FLAG-MSI2 DMSO) 0.0 c d cMYC K562K562 MOLM13MOLM13 DMSO SMAD3 TGFBR1 CDKN1A Ro Up regulated genes p<0.05 Ro Up regulated genes q<0.05 NES 1.68 NES 1.83 Gene sets functional pathways p<0.00001 p<0.0014 MOLM13+Ro K562+Ro (999) 157 (1401)

AML/CML-BC cell lines MSI2 shRNAs AML/CML-BC cell lines MSI2 shRNAs 543 Ro Down regulated genes p<0.05 Ro Down regulated genes q<0.05 NES -1.46 NES -56 175 428 p<0.0045 p<0.00001

AML/ CML-BC cell lines AML/CML-BC cell lines MSI2 shRNAs AML/CML-BC cell lines MSI2 shRNAs +MSI2 shRNAs (1996)

e f g h K562 MOLM13 K562 MOLM13

TGFBR1 TGFBR1 TGFBR1 TGFBR1

cMYC cMYC cMYC cMYC MSI2 MSI2 HOXA9 HOXA9 SMAD3 MSI2 SMAD3 MSI2 ACTIN SMAD3 P21 SMAD3 P21 P21 ACTIN P21 ACTIN ACTIN ACTIN bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6 a 10,000 MLL-AF9 GFP+ DMSO [Secondary BM cells] Ro 4h and 12h

Sacrifice for analysis C57BL/6J 3 weeks Surface flow Sublethally 13.75 mg/kg IC flow irradiated Ro 08-2750 Organ weight IP injection b Surface flow c IC flow 1.50 ** * 1.75 ** 1.25 1.50 **

1.00 1.25

1.00 0.75 0.75 (MFI, normalized) (MFI, normalized) 0.50 0.50 cKit

0.25 cMYC 0.25

0.00 0.00

Ro 4h DMSO Ro 4h DMSO Ro 12h d Ro 12h 10,000 MLL-AF9 GFP+ Day 1 Day 4 Day 7 Day 10 Day 13 [Secondary BM cells]

Sacrifice for analysis 3 days 13.75 mg/kg Monitor Weight Loss C57BL/6J Ro 08-2750 19 DAYS from BMT Sublethally IP injection [72h after last round] irradiated

Spleen WBC IC flow e f 350 * 0.4 * g 100 * 300 80 0.3 250 L) µ 200 60 (K/

0.2 (% freq) 150 WBC

Weight (g) 40 100 0.1 cMYC 20 50

0.0 0 0

DMSO DMSO DMSO

Ro 13.75 mg/kg Ro 13.75 mg/kg Ro 13.75 mg/kg bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

22 24 30 62 64 66 89 92 94 97 I I I I I I I I I I MSI2_RRM1 22 KMFIGGLSWQTSPDSLRDYFSKFGE-IRECMVMRDPTT-KRSRGFGFVTFA-DPASVDKVLGQPHHELDSKTIDPKVAFPRRAQPKMVTRTKK 112 MSI1_RRM1 21 KMFIGGLSWQTTQEGLREYFGQFGE-VKECLVMRDPLT-KRSRGFGFVTFM-DQAGVDKVLAQSRHELDSKTIDPKVAFPRRAQPKMVTRTKK 111 SYNCRIP_RRM1 163 EIFVGKIPRDLFEDELVPLFEKAGP-IWDLRLMMDPLT-GLNRGYAFVTFCTKEAAQEAVKLYNNHEIRSGKHIGVCISVANN 243 HUR_RRM1 21 NLIVNYLPQNMTQDELRSLFSSIGE-VESAKLIRDKVA-GHSLGYGFVNYVTAKDAERAINTLNGLRLQSKTI--KVSYARP 98

111 113 151 153 155 178 181 183 I I I I I I I I Extended Data Figure 1 MSI2_RRM2 111 KIFVGGLSANTVVEDVKQYFEQFGK-VEDAMLMF------DKTT-NRHRGFGFVTFE-NEDVVEKVCEIHFHEINNKMVECKKAQPKEV 190 MSI1_RRM2 110 KIFVGGLSVNTTVEDVKQYFEQFGK-VDDAMLMF------DKTT-NRHRGFGFVTFE-SEDIVEKVCEIHFHEINNKMVECKKAQPKEV 189 SYNCRIP_RRM2 243 RLFVGSIPKSKTKE---QILEEFSK-VTEGLTDVILYHQPDDKKK---NRGFCFLEYEDYHQPDDKKKNRGFCFLEYEDHKTAAQARRRL 306 HUR_RRM2 107 NLYISGLPRTMTQKDVEDMFSRFGR-IINSRVLV------DQTT-GLSRGVAFIRFDKRSEAEEAITSFNGHKPPGSS-EPITVKFAAN 188

RRM1 identities (to RRM1 MSI2): MSI1 73/90 (81%); SYNCRIP 23/90 (26%); HUR 23/90 (26%) RRM2 identities (to RRM2 MSI2): MSI1 75/80 (94%); SYNCRIP 18/80 (23%); HUR 16/80 (20%) a b

110 RRM1 [KD = 36.4 ± 2.1 µM]

100 90 22 24 30 62 64 66 89 92 94 97 I I I I I I I I I I 80 MSI2_RRM1 22 KMFIGGLSWQTSPDSLRDYFSKFGE-IRECMVMRDPTT-KRSRGFGFVTFA-DPASVDKVLGQPHHELDSKTIDPKVAFPRRAQPKMVTRTKK 112 MSI1_RRM1 21 KMFIGGLSWQTTQEGLREYFGQFGE-VKECLVMRDPLT-KRSRGFGFVTFM-DQAGVDKVLAQSRHELDSKTIDPKVAFPRRAQPKMVTRTKK 111 70 SYNCRIP_RRM1 163 EIFVGKIPRDLFEDELVPLFEKAGP-IWDLRLMMDPLT-GLNRGYAFVTFCTKEAAQEAVKLYNNHEIRSGKHIGVCISVANN 243 60 111 113 151 153 155 178 181 183 50 I I I I I I I I MSI2_RRM2 111 KIFVGGLSANTVVEDVKQYFEQFGK-VEDAMLMF------DKTT-NRHRGFGFVTFE-NEDVVEKVCEIHFHEINNKMVECKKAQPKEV 190 40 MSI1_RRM2 110 KIFVGGLSVNTTVEDVKQYFEQFGK-VDDAMLMF------DKTT-NRHRGFGFVTFE-SEDIVEKVCEIHFHEINNKMVECKKAQPKEV 189 SYNCRIP_RRM2 243 RLFVGSIPKSKTKE---QILEEFSK-VTEGLTDVILYHQPDDKKK---NRGFCFLEYEDYHQPDDKKKNRGFCFLEYEDHKTAAQARRRL 306

Fraction bound (%) 30

20 10 RRM1 identities (to RRM1 MSI2): MSI1 73/90 (81%); SYNCRIP 23/90 (26%) RRM2 identities (to RRM2 MSI2): MSI1 75/80 (94%); SYNCRIP 18/80 (23%) 0 10-1 100 101 102 103

Ro 08-2750 (µM) bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Extended Data Figure 2

a b RNA binding

125 WT [KD = 6.7 ± 1.7 µM] F97 F97A [KD = 6.1 ± 0.7 µM] F66A/F97A/R100A [KD > 50 µM] 100 (Ro) 75 R100 3.0 3.5 50 Fraction bound (%)

Ro 08-2750 25 3.1 0

F66 10-2 10-1 100 101 102 RNA (µM) c d Fluorescence Polarization 350

300 (mP) 250 O OH N N O N N O 200 H LiBH4 NH 0 - 22oC, MeOH NH 150 N N IC50 = 2.0±0.3 µM IC50 = 25.0±8.0 µM O O 100 Ro Ro Ro-OH 50 Ro-OH Fluorescence polarization Ro-NGF 0 10-2 10-1 100 101 102 Compound (µM)

e f

F97 F97

3.0 R100 R100 3.4 2.5 3.1 Ro-NGF Ro-OH

3.7 3.6 F66 F66

g h Ro-OH Ro-NGF bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Extended Data Figure 3 Ro-OH

2017-111.10.fid — Group Derek Tan — .* rotsidec 2017-111 (10 1) DMSO 24.0C May_24,2017_17:07:35 Bruker AVIII 600MHz DCH cryo ZRC 2134: zg30 : 1H 6.175 ppm *. — :Proton DMSO /opt/users/rotsidec rotsidec 90

a 0 5 5 0 3 4 1 6 9 7 5 4 7 1 8 7 5 2 5 6 3 8 8 3 3 7 8 9 6 0 3 . 3 9 7 7 6 6 6 8 9 ...... 1 2 3 4 4 5 5 5 7 7 1 H) spectrum 1 NMR ( 0 0 1 0 4 8 6 7 7 8 0 9 7 8 ...... 2 2 1 1 0 0 0

11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm)

2017-111.12.fid — Group Derek Tan — .* rotsidec 2017-111 (12 1) DMSO 24.0C May_24,2017_17:15:06 Bruker AVIII 600MHz DCH cryo ZRC 2134: zgpg30 : 13C 130.000 ppm; 1H 4.000 ppm *. — :Carbon DMSO /opt/users/rotsidec rotsidec 90 7 6 6 3 2 4 1 4 5 2 4 2 8 8 5 0 3 2 5 5 7 0 b 5 2 1 0 3 8 8 6 3 3 2 8 8 5 8 4 5 7 3 5 5 5 0 3 7 9 1 ...... 7 6 2 9 5 0 9 7 3 3 1 1 2 . . . 5 5 5 4 3 3 3 3 3 1 0 1 7 1 1 1 1 1 1 1 1 1 1 6 3 1 C) spectrum 13 NMR (

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Extended Data Figure 4

Supplemental Figure 7 a Ro Ro-OH Ro-NGF c Ro A Ro 08-2750 Ro-OH Ro-NGF

Supplemental Figure 7 A

B b Complex Apo Ro 08Ro-2750 RoRo-OH-OH Ro-NGFRo-NGF B Complex Apo

C C D Ro 08-2750D Ro 08-2750

d Ro-OH E RoOH

E RoOH bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

05-Mar-2018 11:40:09 Muse Annexin V & Dead Cell 1.4 05-Mar-2018 11:46:03 05-Mar-2018 11:47:20 Muse Annexin V & Dead Cell 1.4 Muse Annexin V & Dead Cell 1.4 Acquisition Date and Time : 26-JAN-2017 16:35:23 Acquisition Date and Time : 26-JAAcNq-u2i0s1it7io n2 D0:a4t4e: 1a5nd Time : 26-JAN-2017 20:23:12 Analysis Date and Time : 26-JAN-2017 16:38:20 Analysis Date and Time : 26-JAAnNa-l2ys0i1s7 D 2a0t:e4 5a:n5d5 Time : 26-JAN-2017 20:32:33 Instrument Serial Number : 7200120422 Instrument Serial Number : 7200In1s2tr0u4m2e2nt Serial Number : 7200120422 FileName : MLLAF9 Ro 5-10 4-8h TREATM FileName : MLLFAiFle9N Ramo e5 -:1 0 4-8h TREATM MLLAF9 Ro 5-10 4-8h TREATM Sample # : 7 Sample # : 33 Sample # : 21 Sample ID : 769 LC DMSO Sample ID : 137MSaSmI2p Rleo 1ID0 :8 h 774-3 Ro10 8h 05-Mar-2018 11:43:44 05-Mar-2018 14:30:03 Muse0 A5-nMnaer-x2i0n1 V8 &14 :D30e:a33d Cell 1.4Muse Annexin V & Dead Cell 1.4 Muse Annexin V & Dead Cell 1.4 Annotation : ExtendedAnn oDatatation : FigureA 5nnotation : Acquisition Date and Time : 26-JANcq-2u0is1it7io n1 6D:a4t7e: 0a9nd Time : 27-JAANc-2q0ui1s7it i o1n1 :D04a:t5e7 and Time : 27-JAN-2017 11:05:50 Analysis Date and Time : 26-JANna-2ly0s1is7 D 1a6t:e5 8a:n5d5 Time : 27-JAANn-2a0ly1s7is 1D1a:0te4 :a5n7d Time : 27-JAN-2017 11:05:50 DMSO Ro 5 µM Ro 10 µM Instrument Serial Number : 7200In1s2t0ru4m2e2nt Serial Number : 72001In2s0tr4u2m2ent Serial Number : 7200120422 FileName : MLLAFiFle9N Raom 5e- 1: 0 4-8h TREATM16h apFoilepNtoasmise M : LLAF9 16h apoptosis MLLAF9 Sample # : 13 Sample # : 2 Sample # : 3 Sample ID : bad Sample ID : SampSlea_m00p2le ID : Sample_003 05-Mar-2018 14:51:30 8h Muse Annexin V & Dead Cell 1.4 Muse Annexin V & Dead Cell 1.4 Annotation : 05-AMnanr-o20ta18ti o 1n4 ::4 9:30 05-MAanr-n20o1ta8 t i1o4n:2 :8 :48 Muse Annexin V & Dead Cell 1.4

Acquisition Date and Time : 16-DEACcq-2u0is1it6io n1 7D:2a9te:3 a2nd Time : 23-JAANc-q2u0i1s7iti o 1n6 D:3a6t:e2 9and Time : 23-JAN-2017 16:36:59 Analysis Date and Time : 26-FEABn-a2ly0s1is8 D10a:t3e5 a:5n3d Time : 26-FEABn-a2ly0s1i8s D10a:t3e2 a:4n6d Time : 26-FEB-2018 10:33:20 Instrument Serial Number : 72001In2s0tr4u2m2ent Serial Number : 72001In2s0tr4u2m2ent Serial Number : 7200120422 FileName : GST_F1i6leDNEaCm2e0 :1 6_172807 GST_F2i3leJNAaNm2e0 1: 7_163136 GST_23JAN2017_163136 Cell Conc. % Gated Cell Conc. % Gated Sample # : 2 SCamelpl Cleo #n :c . % Gated8 Sa(Cmepllse /# m : L) 9 (Cells / mL) 05-Mar-20S1a8m 1p1l:e5 9ID:5 0: S05a-MmapSr-le(2aC0_m1e08p0l lls 2e1 3 /I :Dm49 L:: 2)7 MusSea Amnpnle_xi0n0 V8 & Dead Cell 1.M4use Annexin V & Dead Cell 1.4 16h 05-MSara-2m01p8l e 1 I3D:4 :6 :48 Sample_009 Muse Annexin V & Dead Cell 1.4 Annotation : Annotation : Annotation : Live (LL) : Live 3(L.4L4) E: +05 81.85 % 2.13E+05 57.80 % AcLqivueis (iLtioL)n :D ate and Time : 23-JAANc-q2u20i.1s8i79ti Eo 1n+6 0D:53a2te:3 a4nd T8i1m.7e8 : %16-DEACcq-2u0is1it6io n1 4D:2a8te:2 a4nd Time : 16-DEC-2016 14:27:32 Early Apoptotic (LR) : Early2 A.3p8oEp+to0t4ic (LR) : 5.65 % 3.99E+04 10.85 % AnEaalyrslyi sA pDoapteto aticn d(L TRim) :e : 26-FEABn-a2l2y0.s51is18 E D1+a00t:e24 5a:n0d1 Tim7e.1 :1 %05-MAARna-2ly0s1is8 D1a3t:e4 9a:n0d3 Time : 05-MAR-2018 13:46:17 Late Apop./ Dead (UR) : Late 5A.1p3oEp.+/ 0D4ead (UR1)2 : .20 % 1.14E+05 31.00 % InsLtrautem Aenpto Sp.e/ rDiael aNdu (mUbRe) r: : 72001In2s0tr4u32m.92e0nEt +S0e4rial Num1b1e.r0 :4 %72001In2s0tr4u2m2ent Serial Number : 7200120422 Debris (UL) : Debr1is.2 (6UEL+) 0: 3 0.30 % 1.29E+03 0.35 % FileName : GST_F2i3leJNAaNm2e0 :1 7_163136 GST_F1i6leDNEaCm2e0 :1 6_142242 GST_16DEC2016_142242 Debris (UL) : Tota2l .A35pEop+t0o2tic : 0.07 % 7.51E+04 17.85 % Sample # : 1 Sample # : 3 Total Apoptotic : 1.54E+05 41.85 % Total Apoptotic : C6e.l4l C1Eo+nc0.4 %1 G8.a1t5e d% SaCmelpl lCe o#n :c . % Gated2 Cell Conc. % Gated Sample ID : SampSlea_(mC0p0el1ells I D/ m : L) Sample(_C0e0ll3s / mL) (Cells / mL) 24h Sample ID : Sample_002 Annotation : Annotation : Annotation : Live (LL) : Live2 (.L1L9)E : +05 83.35 % Live5 .(5L4LE) +: 05 71.10 % 2.34E+05 45.00 % Early Apoptotic (LR) : Early2 .A2p4oEp+t0ot4ic (LR) : 8.50 % Ear7ly.0 A1pEo+p0to4tic (LR) 9: .00 % 5.32E+04 10.25 % Late Apop./ Dead (UR) : 2.12E+04 8.05 % Reviewed by ______Late Apop./ Dead (UR) : Lat1e. 5A3pEo+p0./ 5Dead (U1R9) .:6 5 % 2.29E+05 4R4ev.1ie5w e%d by ______Debris (UL) : 2.63E+02 0R.1ev0ie w%ed by ______Page 1 Debris (UL) : De1b.r9is5 (EU+L0)3 : 0.25 % 3.11E+03 0.60 % Page 1 Total Apoptotic : TotaC4l eA.3lpl 6CoEpo+tno0ct4.ic : %1 G6.a5t5e d% ToCta2el. 2lAl 3CpEo+pn0tco5.tic : P%ag2 e8G 1.a6t5e d% C2e.l8l C2Eon+c0.5 %5 G4.a4t0e d% (Cells / mL) (Cells / mL) (Cells / mL) 48h Live (LL) : Live1 .(5L2LE) :+ 06 77.15 % Live4 .(9L2LE) +: 05 48.20 % 2.87E+05 33.25 % Early Apoptotic (LR) : Ear2ly. 9A8pEo+p0to5tic (LR) :1 5.15 % Ear2ly. 4A2pEo+p0to5tic (LR) 2: 3.65 % 2.17E+05 25.05 % Late Apop./ Dead (UR) : Late1 .A4p6oEp+.0/ 5Dead (UR7.)4 :5 % Late2 .A8p5Eop+.0/ 5Dead (U2R7) .:9 0 % 3.56E+05 41.20 % Reviewed by ______Debris (UL) : Deb4r.i9s1 (EU+L0) 3: 0.25 % Deb2r.i5s5 (EU+L0) 3: 0R.2ev5ie w%ed by ______4_._3_2__E__+_0__3______0_R._5ev0ie %wed by ______Page 1 Total Apoptotic : Tota4l .A44pEop+t0o5tic : 22.60 % Tota5l .A27pEop+t0o5tic : 51.55 % 5.73E+05 Page6 16.25 % Page 1 Cell Conc. % Gated Cell Conc. % Gated Cell Conc. % Gated (Cells / mL) (Cells / mL) (Cells / mL)

Live (LL) : Live9 .(2L4LE) :+ 05 70.50 % Live6 .(2L3LE) +: 05 57.99 % 2.82E+04 9.20 % Early Apoptotic (LR) : Ear2ly. 1A2pEo+pt0o5tic (LR) : 16.15 % Ear2ly.4 A1pEo+p0to5tic (LR) 2: 2.41 % 8.33E+04 27.15 % Late Apop./ Dead (UR) : 2.01E+05 18.76 % Reviewed by ______Late Apop./ Dead (UR) : 1.67E+05 12Re.v7ie5w e%d by __L__a_t_e_ _A__p_o__p_._/_ D__e__a_d__ _(U__R__R)_ e:v iewed by ______1_._9_4__E__+_0__5______6_3_.31 % Debris (UL) : 8.90E+03 0.83 % Page 1 Debris (UL) : 7.87E+03 0.60 % Debris (UL) : Page 1 1.07E+03 Page0 1.35 % Total Apoptotic : Tota3l .A79poEp+t0o5tic : 28.90 % Tota4l. 4A2pEop+t0o5tic : 41.18 % 2.78E+05 90.45 %

Reviewed by ______R_eviewed by ______Reviewed by ______Page 1 Page 1 Page 1 0 0 0 6 6 6 - - M - M M A A A S S F I T D L E L A S S F I A A T D L E L n a A S S F I A A T D L E L n a o n a o a i i a i r n a a s n c a o i e r n a a s n c a v a i e l i v - r n a a s n c a l t e - e v t t l e 2 a q n t - t m m a r e t 2 n t a q b e m m a r e e t b r 2 n t a q e m m a r e r l b 0 N e l o r 0 N u l u y o l l u l u y r 0 N l y

o r

u l u y y

1 p p ( l

A r 1 p p ( A i y

A i t m

A i a 1 p p ( t m i s A L A a s A i s A L 8 a s t m i s a l l 8 a s s A L s l l 8 a s e e p i m p l l

L

e e p i m i p t e p

L

e e p s i m i p

( t e p

L t s i i

( t e p

o t s 0 o i

( i U ) o t o 0 o i I # n o i U )

o e o 0 o o I # n o i U ) D

e o 9 D o I # n o p D

e p 9 D o : p D

n t p 9 D

L p : p

n t p : n

L p :

n t : : n

L p : .

3

t : : n

S a : .

3 t :

) / t S a : . : o

2 t :

) / t S a : o

3 o :

) / t D

: o

8 o : t D

D

7 o e : t D

t D

: e e : t t D t : e e : i t 2 t : e i a i 1 t r c i a i 2

e r c c a i 2 e r c i c a 4

e i c t

a 8 a i t

a a a e : t a a e : n ( a

e : n ( l

n ( d L l

d L l d

d L N a d

N a d R N a R

( R n

( u T n

( U u T n ) U u T d ) U m i d )

m i d

m m i R :

m

R : m

R : T

b T

b T ) e b ) i e )

e i e m i

e :

m

e m :

: :

r

:

r

: r e

e : e : 0

:

0

0 :

6 :

: 6

6 m 1 G 7 0 1

- S 2 G 7 0 1 - S 1 a 7 0 0 M - M 2 6 6 M 2 6 5 A S S F I A A T D L E L S o p 2 5 2 a S n A S S F I A A T D L E L a a A S S F I A A T D L E L n 0 - - o a n 0 - - a i a i o l o 0 - - m a n a a s n c r T M F e o a i m v i m T M D l a i a n a a s n c r - i e 0 M F t r n a a s n c a v 96h Extended DataFigure6 e e 72h 48h c a l 0 t v - n t a q 2 l t p 0 a r e m m - e _ b t t E e e n t a q 2 _ r t a r e m m p 2 n t a q b 1 A l m m a r e e p E E N 0 b r 1 1 A o e y u l u r l 1 A l N 0 1 r l o

y 0 N y u l u 1

o l B M l ( p p 1 u l u y r 2 A l

y l r A i

3 R e 2 y

t m i ( p p 1 B C

a A e 2 R 1 p p ( A L s A i ( C 6 A s t m i R A i a s 8 a 5 ( C t m i A L s a l l s 0 ( C s A L a s 8 C - s . _ 0 8 s a p e e l l O C p m i - _ 0 l l L

F C - i p e e 1 0 6 4 1 1 2 - - p t e p m i e e p D s L

e - m i p 3 1 1 2 1 (

i 4 2

L t p t e 5 8 3 2 1 2 i e i s t e p 0 4 2 2 o

(

e s o 0 t 0 4 2 e

( U ) i i t o o

L . . . . . E 0 i e o I # n o 0 o e U ) i e l 0 o . . . . . o o 2 0 i U )

D o

I # n . . . . . 0 1 0 0 9 4 d o E l D 9 e l I # n o 0 2 0 0 p o l e 5 5 3 2 1 p

D 0 2 0 l o l : D 9 3 1 1 2 7 Counts Counts D

n t p l M 1 9 D l L p p l p

: l B p

n : n t

C 2 1 : l

L p

1 0 0 1 5 1 s m n t :

l

L p 2 2 1 1 C . :

n :

C 6 0 5 1 4 s t

3

C 1 2 1 : n

S a : 8 s 6 6 4 3

. : 7 : ) / t t

2 : . o : S a 3 t :

8 S a - 7 ) / t

E E E E E 2

o 3 o :

) / t 8 6

D : o

: o E E E E E

8 / : t E E E E E o 7 s D o K

D

9 o : e 2 / o

D

t / t

e : D

t

: e 0 D t t e : i

e : + + + + + m n t 5

o 0 : e

t 1 i a + + + + + m n i t c r 0 0 n + + + + + m 5 i 5 1 1 e

1 i a c 1 c r a i 0 1 c r c i 0 0 0 0 0 e

a 9 c e c 4 9 1 c

t 0 0 0 0 0 c i a 1 9 0 0 0 0 0 L a i 6 2 1 a a

t : e L . 7 a t

3 L a 5 0 4 4 6 a ( n . : : e a

. : e : 5 3 5 5 6 l 6 : 5 3 5 5 6 ) ( n 2 : : 3

n ( d L 3

) _ l 3 : ) l d 0 2 d L d L N a

4 d _ d

2 N a R 3 N a 1 7

9 6 0 R ( R n 7

u T 1 (

: n ( U : n 4 u T ) : T u 6 : : 2 U d 0 U ) m i 3 : )

4 1 3 d d m i m 2 3

m i R :

h 7

6 m R : m

8 R : 1 5 4

T MOLM13

b 9 1 CD14

T bioRxiv preprint T ) MOLM13 b e b i 4 ) % e )

e e 5 i m % CD13 i %

: e

m e m 5 :

: : 7 0 3 3 9

r 5

DMSO 2 0 9 1 7 :

2 0 1 9 7

r : r e 6

G R

. . . . 2 e : e R G G R 3 . . 4 6 3 . 3 . 6 2 0 9 2 :

:

e 1 0 3 5 e .

e :

a . . . v . . . 7 3 0 6 7 : a M : a v v 7 0 0 7 1 0 5 5 9 5 M

M 1 1 S 2 G 7 i

t e - i S 3 a 7 0 0 i 7 S 4 G 7 0 1 e t t e e 6 6 4 9 0 6 % % % % w 2 6 6 e u APC e a w % % % % w S u - p 2 2 2 u

2 6 5 a d a

S

% s 0 - - d e d APC m s % % % % % % s e o 0 - - e 0 - - T F F m d m e F F 0 d T M D d e e

p 0 0 _ p E E b

1 b _ p E E b A

p

y E 1 A 1 A A 1 l y y M B B e 2

1 l l B B _ n

e 2 e 2 ( C 6 C n _ R _ n ( C _ 0 _ 5 ( C C - - MOLM13 n O _ _ _ 0 _ 0 C - - F n C n 2 2 1 8 5 2 2 _ e - - 0 4 _ D _ 3 5 2 1 2 2 2 e 5 1 2 2 5 e _ e 0 4 e e 0 4 2 2 e L . . . . . 0 0 E _ _ e l e 0 2 _ . . . . . 0 0 7 1 8 4 9 x . . . . . l l _ E l _ 0 2 l x 0 2 0 0 x 7 8 4 3 6

2 7 3 9 5 l M 1 1 _ l l l B i l 0

C 2 2 _

_ 1 1 0 6 5 9 0 s 0 l i l i _ n 0 C 3 2 4 2 1 1 C 9 6 4 6 6 s C 3 3 0 3 4 s 7 7 n _ _ n 6 6

_ - 7 7 E E E E E 2 6

o _ _

/ 8 6

- E E E E E

E E E E E

K

_ V - o o /

2 / - M V _ _ V

0 1 M

+ m n + + + + _

M

1 1 doi: _ _ A S S F I A A T D L E L + + + + + m n + + + + + m n 5

0 0 T D L E L A S S F I A A 1 _ n 4 1 T D L E L A S S F I A A a c

n 0 0 0 0 0 & 1 _ _ a 1 n o 4 a c _ c & & o a i 0 0 0 0 0 L 0 0 0 0 0 i 6 1 1 9 o a i r n a a s n c a _ _ i e . 7 : a i a n a a s n c r v L L i e l 5 3 5 4 5 _ 3 a n a a s n c r v certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. - : e l t . 3 :

. v - e _ _ l t t 5 3 5 5 5 ) 5 3 5 5 5 - e 2 n t a q 2 2 : 6 :

t t m m a r e 3 _ D e n t a q 2 b _ t a r e m m e ) ) n t a q 2 b r 2 3 : _ D D _ a r e m m e b r l 3 e 0 N _ r l o 4 N 0 u l u y 1 _ l o l 3 _ _ N 0 y u l u r 1 o l y

y u l u r 9 1

_ e 5 l

y 1 p p ( r

A :

y ( p p 1 A i _ e e _

A t m i 9 ( p p 1 A i : a 1 A t m i https://doi.org/10.1101/321174 s A L 5 : _ A i a s 4 m i t A L s 8 a s a s 6 5 : a : _ _ A L s a s 8 l l 5 s a s 8 l l _ a a 5 e e p 3 : l l m i p 3 p e e

L 6 _ _ p m i i 3 p e e L

t e p 3 p m i i h 0 s _ d L p t e

( 6 i s t p t e (

i _ d d _ s t 0

o 8 (

i 0 o 4 Ro 20 t o

i U ) _ i o 0 o 1 o

U ) i I # n o 1 o 0 o e

_ _ U ) i o I # n o o e D

o # n I o 9 D _

e

D p C o D 9 p % 4

D p : _ _ D 9 p n t 5 C C p :

L p p n t %

_ % : L p

: n

t n

L p

n : : _ _

: .

n : : 4

t 5

. : S a _ 3 0 2 9 6 : t

4

e 5 . : S a

) / t t

3

: o _ _ S a

) / t

5 0 3 2 4 : P

4 0 2 2 5 e e o : ) / t

: _ 5 o o

: D

P P : o 1

6

G R

D t 1 . 1 . 8 _ _ o 1 a l D

D

t e :

D _ G R t R G t : e l 8 . 7 0 0 : e 8 . 1 7 1 a l 2 8 D l a t : e e : t _ e t _ g

i l e : l t 9 1 1 CD235a i _ . . . t 1 CD71 e e g

a i i g 1 r c a 2 i a _ v _ c r e ......

e 3 5 4 5 4 i a 1 c M c r 1 e

_ a a 5 c 1 e v v i e e 5 6 3 2 3 2 0 4 8 8 a c i _ M M i 7 _ a

t

i . t e a a

t _ a 1 i i 0 5 5 a

t

. e : a . a 4 _ e t t e e _ : e 1 a 1 0 0 0 4 5 0 5 5 % % w n ( : e 4 _ u

4 ( n K562 e e

l ( n _ w % % w

_ l u u

d L

l K562 d L d

d d L

% % % s

d e N a d d d N a s % % % % % % % % s N a e e R R d e

R ( µ

n ( d d

n e e u T (

n u T U u T U b )

U ) d ) b d A b m i

-

d m i y

M m i A A m

R : y m y R :

m

FITC R :

_ n T

T b T n _ b _ n ) b _ e ) e n i ) - e _ i _

e i n

_ n m e

m : e

m _ : e _ PE

: _

: e

e r :

_ r _ :

r _

x e

e _ _ e x x : _ : 0 i :

0 _ _

i

_ n i

0

: 6 n _ : _ n 6 : _

6

- _ _ k 1 m 7 0 2 -

_ V S 7 G 7 0 1

5 a 7 0 0 S M - M V _ _ V _ 2 M 0 2 6 5 T D L E L A S S F I A A 2 6 9 _ _ S F I A A A S T D L E L p 2 2 2

a o n _ S a a n T D L E L A S S F I A A a

&

o _ _ 0 - - n o a i a _ 0 - - i & & d a i o 0 - - a n a a s n c r m l i e o M J r a s n c n a a m v _ _ e l a i T M D m v - i _ l F F t a n a a s n c r 0 - e e t t v 0 e n t a q 2 _ l _ m t p 0 a r e m m - A 2 t a q n

b t _ D

m m a r e e e b r t _ e n t a q 2 p r D _ l a r e m m _ D 1 A E E p N 0 b E l o e _ 1 A 1 0 N y u l u r o 1 l 96h 72h 48h u l u y r l l _

y N 0 _ N r

o 1 s M y

( p p 1 y u l u _ e l

A l 2 l 1 p p ( A i r A t m i

y e 2 3 R B B A i e _ a

C _ e 2 e m i t A L s ( p p 1 R a s A _ ( C s A L a s 8 A i o s t m i ( C 9 8 s a l l a a - ( C _ A L s _ 0 l l s C p e e a s 8 _ _ 0 _ O a p m i C a 0 _ e e p L

l l - 2 C - - m i p i

L _ p t e - - _ 1 1 6 6 9 i s p e e D e t p ( p m i _ d 1 0 1 8 6 e s t L

4 2 3 4 1 1 1 2 2

( i i e t o p t e 0 4 2 2 k e d _ i o 0 s _ d 4 0

o U ) i (

0 e 0 o o t _ L i U ) o I # n i e . . . . . o e o

e # n I o o o 0

. . . . . l _ e

D U ) i _ 2 0 . . . . . 0 0 o D 9 o l E 5 2 6 1 1 7 D

p o I # n _ 0 2 0 0 l l

9 D p e C 9 0 1 2 3

2 0 p : o l 1 l 4 2 9 4 4 p n t

D

l M _ : L p

D 9 l _ C t n p

C l l

L p

n : p _

: l 1 1 2 1 C : n

: n t l 6 3 5 0 6 5 s

. : L p

1 C 7 2 1 1 _ : t

4

3 0 1 5 s C _ C 2 5 : . S a

n : 1 0 7 5 0 s 7 4 t

_

S a

) / t

e :

o : . :

) / t : b t

2 _

: o P S a _ e - 7 7 8

: o 6

e 2 E E E E E

D _ ) / t

E 8 6 2 o o : E E E E P

o D

t : P E E E E E

/

D o _ K t : e a l o 9 / 2 _ o D t

D / e : e : _

t l t l a

: e t D a l

i : e

_

t 3 g t _ l 1 + + + + + m n i e : + l 1 0 1 i a _ + + + + m n 7 c r t 0 0 g + + + + + m n a i 1 i g

5 r c e

5 _ 1 c e _

e 0 i a 1 1 c i _ c r 1 c 8 a e 1 8 0 0 0 0 0 i e

0 c e 9 a

t c 0 0 0 0 _ c 2 a

1 1 9 _ 0 0 0 0 0 t

. i 6 1 L a a _ a : e 1 L

a

t Fold change . 9

L . e : 4 a _ . ; ( n 1 . _ : : 5 3 4 4 5 a 5 1 . : n ( _ : e 4 5 0 4 5 h

l 6 : 4 5 4 5 5 6 2 2 : 1 ) l d L ( n _ 4

) _ d L 4 : )

d l 2 d N a d L

this versionpostedMay14,2018.

3 N a _ d 3 3 R r 5 N a R 0

9 3 (

n R o ( 2 n u T b

: : u T U ( : n U ) : u T 6 3 : 2 )

d (MFI, normalized) 0 U 1 d m i 5 :

) m 3 m i

d m 4 R : m i m

h 7 5 R :

1 0

m 1 R : 7

T

T b 9

_ b ) T e - ) b e i 1.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 i

) % e e 2

m % M e i % m :

: e

m

: :

r

0 1 0 5 5 8 : r 2 0 1 9 7

1 2 1 8 7 :

e r O

e

R 1 G : . . . 8 e G R : 1 G R 3 . 3 . 6 9 . . 8

DMSO 1 5 6

:

0 9 e

3 1 e L . : e .

: . . . . a . . v 1 a 5 5 0 7 : a v M

0 v 2 0 3 2 7 1 5 0 5 M m 4 m 7 2 2 M M

i S 5 G 7 0 1 i e t i 5 MOLM13 S 1 a 7 0 0 t e 0 t e 0 7 6 3 e 0 5 2 5 5 e w % % % e u 2 6 9 % % w % % w u a 2 o u _

S 4 p 2 2 2

a

d

% 0 - - d s % d e 0 - - % % % % s % % s e m l e K o 0 - - J J T M D CD13 m m d d e 0 d F F d e 0 e A A

p 0 5 _

b p m

1 b E b

1 A p E E 1

A A y 1 A N N y _ 1 y l M 2

e 2 l 3 C s

R B B

n _ e 2 ( C _ A n DMSO 9 ( C _ n - - 0 ( C C _ _ 0 o _ C _ n O 2 2 _ _ 0 n - - C - - 4 6 2 1 9 n D _ 7 3 5 2 1 e P 4 _ e _ 0 4 2 2 4 4 1 3 3 2 2 k e e e 0 0 e _ 0 4 e * e . . . . . _ L _ . . . . . l e 2 E l 3 7 8 4 1 5 O _ 0 2 0 0 . . . . . 0 0 l x 8 1 4 3 1 _ l l l 1 1 _ x 1 2

x l 9 0 6 2 1 l M l _ K562 l l C 2 _ i

2 4 5 7 5 s 6 _ 5 2 1 1 C 1 1 i C 0 7 5 5 5 s l i 7 7 P n _ 20 µM Ro C 4 2 4 3 9 5 2 s _ n _ n

E E E E E 2 _ 8 6

- 7 7 E E E E E o _

_ /

o

2 / E E E E E

2 K V _ o

1 / _ V

_ V

+ + + + + m n 1

_ + + + + + m n 7 0 0 1 _ 4 _ + + + + + m n 8 5

1 1 _ c

0 0 0 0 0 8 _ c _ & 0 0 0 0 0 2 1 9 3 H & c & L _ 0 0 0 0 0 : L 6 _ 9 3 _ . 1.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 . L 5 2 5 5 5 0 : _ 5 3 5 5 6 h 6 : . : _

_ ) 0 5 2 5 5 5

) 2

4 : 5 : D _ ) 7 _ D _ D

3 5 _ 7 _ r

_ 2 6 _ : 9 o e _ 1 6 b _ e _ e 5 : : : _ 5 _ 6 _ 1 a 0 : 0 : m a a 1 _ 2 0 _ _ 1 h 0 d _ 1 6 _ d _ d 0 6 _ _ _ _ -

_ % C 2 _ _ M % C Ro 20 C % _ _ _ 3 0 2 1 6 0 _ 4 0 2 1 5 CD71 e _ _ K562

e e 6 0 2 4 3 O P

P _ P G R

2 . 1 0 7 _ _ R G 0 . 8 2 9 l a G R 0 a l _ a l 1 . 0 0 8 l 1 e _ _ l l e L g 0 . . . . g

_ g e . . . . a v _ 1 _ a 0 5 1 9 9 . . . . v M 1 e 1 3 6 1 1 5 a M e _ e v M 3 5 9 3 6 i _ _ M DMSO i t e

. e t 5 5 0 0

_ i . 1 . 2 5 7 2 e _ _ t e 1 4 1 e % w 0 5 5 5 u 4 4 w % _ e u _ _ _ % w

u

d d % % % % s *

e s % % % % d e K % % % % s e d e d e The copyrightholderforthispreprint(whichwasnot d e

5 b

b 20 µM Ro

A µ b

A y y _ A y

_ n

n _ A M _ n _ _ n n _ _ n P _ e _ e _ _ e _ O _ x _ x _ x _ _ i i _ P _ n i n _

_ n

_

_

_ 2

_ V

V

_

_

V _

_ 4

_

_

_

&

_ &

H _ &

_ _ _ _

_ _ D

D _ _ D _ _ _ _ e e _ _ e _ _ a _ a _ a _ _ _ d d _ _ d _ _

_

_ C

_ C _ C _ _ _ _ e _ e P _ e P _ _ P a l _ l a _ l _ a l l g

_ l g _ _ 1 g

1 e _ e _ 1 _ e

_ .

_ . 1 _

1 4 . _ 4 1 _ 4 _ _ bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Extended Data Figure 7 a b K562 c MOLM13

2.5 2.0 1.50 DMSO * DMSO * 1.8 Ro 20 µM Ro 20 µM ) 1.25 ) 1.6 2.0 * * ns 1.4 ACTIN 1.00 ACTIN 1.5 1.2 0.75 1.0 1.0 0.8 OP-Puro 0.50 0.6 mRNA expression mRNA Ct, normalized to mRNA expression mRNA Ct, normalized to

ΔΔ 0.4 ( (AF647 MFI, normalized) ΔΔ 0.5 0.25 ** ** ( 0.2

0.00 0.0 0.0 1h 2h 4h

0.5h MSI2 MSI2 cMYC cMYC HOXA9 SMAD3 DMSO SMAD3 TGFBR1 CDKN1A Pre-treatm. TGFBR1 CDKN1A CHX0.5h NoOp-Puro Co-incRo 1h CHX+Ro0.5h bioRxiv preprint doi: https://doi.org/10.1101/321174; this version posted May 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Extended Data Figure 8 a DMSO Ro 13.75 mg/kg 22 22 20 20 18 18 16 16 14 14 12 12 10 10 Sac'd #1 #6

Weight (g) 8

Weight (g) 8 #2 #7 #1 #5 6 6 #3 #8 #2 #6 4 #4 #9 4 #3 #7 2 #5 2 #4 #8 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0 1 2 3 4 5 6 7 8 9 10111213141516171819 Days after BMT Days after BMT b c

ns ns 9 1500 8 1250 7

6 1000 L) L) µ

5 µ

(M/ 750 4 (K/ PLT RBC 3 500

2 250 1

0 0

DMSO DMSO

Ro 13.75 mg/kg Ro 13.75 mg/kg