Cohesin loss and MLL-AF9 are not synthetic lethal in murine hematopoietic stem and progenitor cells

Abstract Objective As cohesin mutations are rarely found in MLL-rearranged acute myeloid leukemias, we investigated the potential synthetic lethality between cohesin mutations and MLL-AF9 using murine hematopoietic stem and progenitor cells. Results Contrary to our hypothesis, a complete loss of Stag2 or haploinsufficiency of Smc3 were well tolerated in MLL-AF9-expressing hematopoietic stem and progenitor cells. Minimal effect of cohesin subunit loss on the in vitro self-renewal of MLL-AF9-expressing cells was observed. Despite the differing mutational landscapes of cohesin-mutated and MLL fusion AMLs, previous studies showed that cohesin and MLL fusion mutations similarly drive abnormal self-renewal through HOXA gene upregulation. The utilization of a similar mechanism suggests that little selective pressure exists for the acquisition of cohesin mutations in AMLs expressing MLL fusions, explaining their lack of co-occurrence. Our results emphasize the importance of using genetic models to test suspected synthetic lethality and suggest that a lack of co-occurrence may instead point to a common mechanism of action between two mutations.


Introduction
Translocations involving the MLL1 gene (KMT2A) are associated with the development of acute myeloid leukemia (AML) and result in the expression of many different MLL fusion proteins, of which MLL-AF9, MLL-AF4, and MLL-ENL are the most common (5).MLL fusions are robust oncogenes and can transform both hematopoietic stem and progenitor cells (HSPCs) and more committed progenitors (5).MLL fusions are found in approximately 5-10% of patients with AML and drive HSPC self-renewal and leukemic transformation (5,6).Despite the large degree of genetic complexity found in AML (7), AMLs with MLL fusions have a small number of co-operating mutations and display a clear preference for the acquisition of RAS family mutations, including KRAS, NRAS, PTPN11, and BRAF (5,6,(8)(9)(10).
Approximately 10% of patients with AML have a mutation in one of four cohesin complex members (STAG2, SMC1A, SMC3, or RAD21) (7,(11)(12)(13)(14)(15). Cohesin mutations are mutually exclusive and result in haploinsu ciency and increased HSPC self-renewal (7,(13)(14)(15)(16).Despite their similar rates of overall occurrence, MLL fusions and cohesin mutations are rarely observed together.The rate of cohesin mutations in all MLL fusion AMLs is approximately 7% (cBioportal OHSU Cancer Cell 2022 and TCGA NEJM 2013 datasets and (6, 8, 17)).The rate of co-occurrence is particularly low in AMLs harboring MLL-AF9 (cBioportal, 2.7% and (8)).Unlike MLL-AF9, cohesin mutations do not often co-occur with RAS family mutations and instead co-occur most often with NPM1 mutations (7,12,13,18).The difference in cooccurrence between cohesin and MLL-AF9 or NPM1 is surprising considering both MLL-AF9 and NPM1driven leukemias are known to be reliant on increased HOXA expression (19)(20)(21)(22).Interestingly, NPM1 mutations are not observed within MLL-AF9 leukemia (10).Due to the low incidence of MLL-AF9 and cohesin mutations in AML and their differing mutational patterns, we hypothesized that cohesin mutations would be detrimental to hematopoietic stem and progenitor cells harboring MLL-AF9.To this end, we expressed MLL-AF9 in HSPCs isolated from mice bearing oxed alleles of the cohesin subunits Stag2 (2) or Smc3 (1) and performed self-renewal assays in excised vs. unexcised cells.In contrast to our hypothesis, we found that both a complete loss of Stag2 as well as haploinsu ciency of Smc3 were well tolerated by cells expressing MLL-AF9.While a signi cant increase in self-renewal was observed with MLL-AF9 expression alone, the addition of a cohesin mutation did not reduce or largely enhance selfrenewal in MLL-AF9 expressing cells.
It is possible that the parental line contained a small number of cells with incompletely excised Stag2 that were selected for with serial passaging.To ensure that full excision of both Stag2 alleles was maintained across all passages, we examined Stag2 excision by PCR, where the unexcised, oxed allele runs at 554 bp and the excised allele runs at 294 bp (Supplemental Fig. 1A) (2).To con rm the sensitivity of our assay, we mixed unexcised Stag2 / and excised Stag2 −/− DNA controls at varying molar ratios and performed the excision PCR.A slight 554 pb band was detectable with a 10% spike-in of unexcised DNA, which was readily detectable at 20% (Supplementary Fig. 1A).No unexcised band was detectable in the parental line (MLL-AF9;Stag2 −/− ) or at any passage (Supplementary Fig. 1A).As exon 7 is excised upon Cre exposure (2), we also performed qPCR using primers spanning the exon 6-7 junction.We readily detected transcript in WT cells, but no transcript was detectable in Stag2 −/− control cells or in our MLL-AF9;Stag2 −/− cells throughout serial passaging, con rming that no read-through was occurring and supporting complete excision (Supplementary Fig. 1B).Western blotting con rmed a lack of Stag2 expression throughout serial passaging (Fig. 1B).Full length blots are presented in Supplementary Fig. 1C.Finally, as Stag2 −/− cells also show enhanced self-renewal, it is possible that MLL-AF9 was lost with serial passaging.However, cells remained GFP + throughout the assay as determined by ow cytometry, indicating retention of the retroviral cassette (Supplementary Fig. 1D-F).We conclude that Stag2 was fully excised, and that this excision was maintained throughout serial passaging.Further, no synthetic lethality was observed between MLL-AF9 and Stag2 in murine HPSCs.

Smc3 haploinsu ciency and MLL-AF9 are not mutually exclusive
In contrast to Stag2, a complete loss of Smc3 is lethal, and AMLs harboring Smc3 mutations are functionally haploinsu cient (1,13).To determine if Smc3 haploinsu ciency is also tolerated in MLL-AF9-expressing HSPCs, we isolated HSPCs from an Smc3 /+ mouse and infected these cells with MLL-AF9-IRES-GFP retrovirus.Following sorting for GFP + cells, cells were transduced with empty vector or tamoxifen-inducible Cre virus.Following 4-OHT treatment of both empty vector and Cre-transduced cells, self-renewal was analyzed by serial replating assays.Similar to Stag2 −/− , Smc3 −/+ HSPCs showed enhanced self-renewal over WT HSPCs (Fig. 2A).MLL-AF9;Smc3 /+ cells (unexcised) also showed enhanced self-renewal, as was observed in our Stag2 assays (Fig. 2A).Excision of one copy of Smc3 in the presence of MLL-AF9 did not result in a loss of self-renewal capacity (Fig. 2A).To check for Smc3 excision, we performed a PCR assay where the excised Smc3 allele is 349 bp, the unexcised, oxed allele is 313 bp, and the WT allele is 287 bp.MLL-AF9;Smc3 −/+ cells maintained excised and heterozygous throughout serial passaging (Supplementary Fig. 2A).Due to the heterozygosity of the cells, we were unable to perform a similar spike-in experiment to check excision sensitivity as was done in Stag2 −/− cells.We performed qPCR for Smc3 and found a consistent 60-70% loss of Smc3 mRNA throughout serial passaging, consistent with excision of one Smc3 allele (Supplementary Fig. 2B).Consistent with our previous results, western blotting also showed a loss of Smc3 protein compared to WT cells throughout serial passaging (Fig. 2B,C)(4).While Smc3 protein levels slightly increased over serial passaging, the level never rose above that observed for our independent Smc3 −/+ control (Fig. 2B,C).
Smc3 protein expression in our MLL-AF9;Smc3 −/+ cells remained similar to that observed in a known Smc3 −/+ control (Fig. 2B,C).As with the Stag2 experiment, cells remained GFP + throughout serial passaging as determined by ow cytometry (Supplementary Fig. 2C,D).Compare to GFP-control Supplementary Fig. 1D).We conclude that a loss of a single allele of Smc3 is compatible with MLL-AF9 in murine HSPCs.

Discussion
In AML, MLL fusions do not co-occur readily with cohesin mutations, but instead co-occur with Ras family mutations (5,6,(8)(9)(10).In contrast, cohesin mutations tend to co-occur with NPM1 mutations but not with Ras family mutations (7,12,13,18).Further, NPM1 mutations are not found in MLL-fusion AMLs (10).Based on these different mutational landscapes, we hypothesized that the addition of cohesin haploinsu ciency to MLL-AF9-expressing HSPCs would be detrimental.Contrary to this hypothesis, we found that both a complete loss of Stag2 and Smc3 haploinsu ciency were well tolerated by MLL-AF9expressing cells.While Stag2 loss increased the self-renewal capacity of MLL-AF9-expressing cells at later passages (Fig. 1A), minimal effects on self-renewal were observed upon excision of one allele of Smc3 (Fig. 2A).Thus, despite their predicted synthetic lethality and differing co-mutational spectra in AML, MLL-AF9 and cohesin mutations are permissible for HSPC growth and self-renewal.
Interestingly, both cohesin haploinsu cient and MLL-AF9 HSPCs and AMLs have elevated expression of HOXA cluster genes, particularly HOXA7 and 9, which are critical for enhanced self-renewal (5,28,29).HOXA9 is also important for AML transformation and maintenance (28, 30,31).Knockdown of HOXA9 reduces proliferation in primary human MLL-fusion AMLs through cell cycle arrest, increased differentiation, and increased apoptosis (28).Over-expression of HOXA9 in hematopoietic stem and progenitor cells results in stem cell expansion and leukemic development, suggesting that both MLL-AF9 and cohesin mutations co-opt key regulators of HOXA gene expression (28).
In support of this, both MLL-AF9 and cohesin-mutated cells in uence gene expression through interactions with the DOT1L and PRC2 histone methyltransferase complexes, respectively (5,29,32).MLL-AF9 recruits the DOT1L methyltransferase to target genes such as HOXA9 (5).DOT1L deposits the activating mark H3K79me2, which drives gene expression.In hematopoietic cells, HOXA7/9 are repressed by the PRC2 complex, which deposits the repressing mark H3K27me3 at promoter regions.The cohesin subunits Rad21 and Smc3 have been shown to interact with components of the PRC2 complex, with cohesin loss resulting in decreased PRC2 recruitment to murine Hoxa7/9 promoters (29).This allows for Dot1L recruitment and enhanced gene activation.Interestingly, DOT1L is critical for both the initiation and maintenance of MLL-AF9 AML, and small molecule inhibitors of DOT1L are particularly effective against mouse and human MLL-rearranged leukemias in vitro and in xenograft models (31)(32)(33)(34).
Furthermore, the abnormal self-renewal observed in murine HSPCs depleted of cohesin were restored by Dot1l inhibition (35).Collectively, these data suggest that DOT1L inhibitors may be particularly effective against MLL-rearranged or cohesin-mutated AMLs.
Because MLL-AF9 is a strong driver of HOXA gene expression and self-renewal and, unlike Rad21depletion, up-regulates the HOXA9 co-factor MEIS1 (28, 29), there is likely little selective pressure for the acquisition of cohesin mutations in MLL-AF9-expressing AMLs, potentially explaining why they are not commonly observed together.Interestingly, NPM1 mutations are more commonly observed with cohesin mutations and, like MLL-AF9, NPM1 also drives HoxA expression.This suggests that additional mechanisms beyond HOXA-induced self-renewal may contribute to NPM1-driven leukemias, and such mechanisms are supported by cohesin haploinsu ciency.We have shown one such mechanism is through Rac-regulated growth and apoptosis (4).While it is important to consider that differences may exist in in vitro vs. in vivo assays as well as between mice and humans, our experiments suggest that the absence of co-occurrence, while often interpreted as synthetic lethality, may instead re ect signi cant overlap in the molecular mechanisms driving the observed disease biology.Intriguingly, both cohesin mutant and MLL-AF9 AMLs are sensitive to DOT1L inhibitors.Thus, a lack of co-occurrence may point to a common, targetable mechanism behind two initially dissimilar appearing leukemias, underlining the importance of genetically testing mutual exclusivity.

Limitations
Assays were performed on HSPCs isolated from a single Stag2 / or Smc3 /+ mouse.Each Stag2 / and Smc3 /+ parental cell line, once infected with MLL-AF9 and treated with 4-OHT, was followed over serial re-passaging, and while some assays were repeated (serial replating), others were not (qPCR, genotyping, western blot, or GFP + assays).Thus, differences may exist between individual mice or with long-term culture, which would not be revealed by our assays.However, the consistency between our results in Stag2 −/− and Smc3 −/+ cells and between passages does not support this.In addition, Fig. 2B and C show that Smc3 protein levels did slightly increase between passage 1 and 4, suggesting a selection for cells with higher Smc3 expression may occur in the presence of MLL-AF9.However, the level of Smc3 expression did not rise above that of an Smc3 −/+ control.Further experiments would need to be performed to determine if higher Smc3 expression is selected for in the presence of MLL-AF9.
Additionally, as mentioned in our discussion section, all of our assays are performed in vitro, and different results may be observed in vivo.Lastly, our experiments focus primarily on leukemogenesis.Thus, loss of Stag2 or Smc3 may contribute to aspects of leukemia maintenance that were not addressed in this manuscript.

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