Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Calcineurin promotes adaptation to chronic stress through two distinct mechanisms.

Adaptation to environmental stress requires coordination between stress-defense programs and cell cycle progression. The immediate response to many stressors has been well characterized, but how cells survive in challenging environments long-term is unknown. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in adaptation to chronic CaCl2 stress in Saccharomyces cerevisiae. We find that prolonged exposure to CaCl2 impairs mitochondrial function and demonstrate that cells respond to this stressor using two CN-dependent mechanisms - one that requires the downstream transcription factor Crz1 and another that is Crz1-independent. Our data indicate that CN maintains cellular fitness by promoting cell cycle progression and preventing CaCl2-induced cell death. When Crz1 is present, transient CN activation suppresses cell death and promotes adaptation despite high levels of mitochondrial loss. However, in the absence of Crz1, prolonged activation of CN prevents mitochondrial loss and further cell death by upregulating glutathione (GSH) biosynthesis genes thereby mitigating damage from reactive oxygen species. These findings illustrate how cells maintain long-term fitness during chronic stress and suggest that CN promotes adaptation in challenging environments by multiple mechanisms.

The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective therapies. Here, we demonstrate that the transcription factor, homeobox C6 (HOXC6), is overexpressed in most PDACs, and its inhibition blocks PDAC tumor growth and metastasis. HOXC6 transcriptionally activates tumor-promoting kinase MSK1 and suppresses tumor-inhibitory protein PPP2R2B in PDAC. HOXC6-induced PPP2R2B suppression causes mammalian target of rapamycin (mTOR) pathway activation, which facilitates PDAC growth. Also, MSK1 upregulation by HOXC6 is necessary for PDAC growth because of its ability to suppress apoptosis via its substrate DDX17. Combinatorial pharmacological inhibition of MSK1 and mTOR potently suppressed PDAC tumor growth and metastasis in PDAC mouse models. PDAC cells with acquired resistance to MSK1/mTOR-inhibitors displayed activated insulin-like growth factor 1 receptor (IGF1R) signaling and were successfully eradicated by IGF1R inhibitor. Furthermore, MEK inhibitor trametinib enhanced the efficacy of dual MSK1 and mTOR inhibition. Collectively, these results identify therapeutic vulnerabilities of PDAC and an approach to overcome acquired drug resistance to prolong therapeutic benefit.

The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing.

Understanding the cell biological mechanisms that enable tumor cells to persist after therapy is necessary to improve the treatment of recurrent disease. Here, we demonstrate that transient receptor potential channel 6 (TRPC6), a channel that mediates calcium entry, contributes to the properties of breast cancer stem cells, including resistance to chemotherapy, and that tumor cells that persist after therapy are dependent on TRPC6. The mechanism involves the ability of TRPC6 to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated calcium entry represses the epithelial splicing factor ESRP1 (epithelial splicing regulatory protein 1), which enables expression of the integrin α6B splice variant. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic inhibition of TRPC6 sensitizes triple-negative breast cancer (TNBC) cells and tumors to chemotherapy by targeting the splicing of α6 integrin mRNA and inducing Myc. These data reveal a Ca2+-dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing.

Nanoparticle delivery of innate immune agonists combines with senescence-inducing agents to mediate T cell control of pancreatic cancer.

Pancreatic ductal adenocarcinoma has quickly risen to become the 3rd leading cause of cancer-related death. This is in part due to its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here we investigated an innovative immunotherapy approach combining local delivery of STING and TLR4 innate immune agonists via lipid-based nanoparticles (NPs) co-encapsulation with senescence-inducing RAS-targeted therapies that can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other pro-inflammatory signaling, increased antigen presentation by tumor cells and antigen presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and Type I interferon-dependent tumor regressions and long-term survival in preclinical PDAC models. STING and TLR4-mediated Type I interferon signaling were also associated with enhanced NK and CD8+ T cell immunity in human PDAC. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can synergize to orchestrate a coordinated innate and adaptive immune assault to overcome immune suppression and activate durable anti-tumor T cell responses against PDAC.

Inflammatory stress-mediated chromatin changes underlie dysfunction in endothelial cells.

Inflammatory stresses underlie endothelial dysfunction and contribute to the development of chronic cardiovascular disorders such as atherosclerosis and vascular fibrosis. The initial transcriptional response of endothelial cells to pro-inflammatory cytokines such as TNF-alpha is well established. However, very few studies uncover the effects of inflammatory stresses on chromatin architecture. We used integrative analysis of ATAC-seq and RNA-seq data to investigate chromatin alterations in human endothelial cells in response to TNF-alpha and febrile-range heat stress exposure. Multi-omics data analysis suggests a correlation between the transcription of stress-related genes and endothelial dysfunction drivers with chromatin regions exhibiting differential accessibility. Moreover, microscopy identified the dynamics in the nuclear organization, specifically, the changes in a subset of heterochromatic nucleoli-associated chromatin domains, the centromeres. Upon inflammatory stress exposure, the centromeres decreased association with nucleoli in a p38-dependent manner and increased the number of transcripts from pericentromeric regions. Overall, we provide two lines of evidence that suggest chromatin alterations in vascular endothelial cells during inflammatory stresses.

Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Therapeutic blocking of VEGF binding to neuropilin-2 diminishes PD-L1 expression to activate antitumor immunity in prostate cancer.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.

Phosphosite Scanning reveals a complex phosphorylation code underlying CDK-dependent activation of Hcm1.

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates Cks1-dependent phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

A brown fat-enriched adipokine Adissp controls adipose thermogenesis and glucose homeostasis.

The signaling mechanisms underlying adipose thermogenesis have not been fully elucidated. Particularly, the involvement of adipokines that are selectively expressed in brown adipose tissue (BAT) and beige adipocytes remains to be investigated. Here we show that a previously uncharacterized adipokine (UPF0687 protein / human C20orf27 homolog) we named as Adissp (Adipose-secreted signaling protein) is a key regulator for white adipose tissue (WAT) thermogenesis and glucose homeostasis. Adissp expression is adipose-specific and highly BAT-enriched, and its secretion is stimulated by ß3-adrenergic activation. Gain-of-functional studies collectively showed that secreted Adissp promotes WAT thermogenesis, improves glucose homeostasis, and protects against obesity. Adipose-specific Adissp knockout mice are defective in WAT browning, and are susceptible to high fat diet-induced obesity and hyperglycemia. Mechanistically, Adissp binds to a putative receptor on adipocyte surface and activates protein kinase A independently of ß-adrenergic signaling. These results establish BAT-enriched Adissp as a major upstream signaling component in thermogenesis and offer a potential avenue for the treatment of obesity and diabetes.

Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.

Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity.

scATACpipe: A nextflow pipeline for comprehensive and reproducible analyses of single cell ATAC-seq data.

Single cell ATAC-seq (scATAC-seq) has become the most widely used method for profiling open chromatin landscape of heterogeneous cell populations at a single-cell resolution. Although numerous software tools and pipelines have been developed, an easy-to-use, scalable, reproducible, and comprehensive pipeline for scATAC-seq data analyses is still lacking. To fill this gap, we developed scATACpipe, a Nextflow pipeline, for performing comprehensive analyses of scATAC-seq data including extensive quality assessment, preprocessing, dimension reduction, clustering, peak calling, differential accessibility inference, integration with scRNA-seq data, transcription factor activity and footprinting analysis, co-accessibility inference, and cell trajectory prediction. scATACpipe enables users to perform the end-to-end analysis of scATAC-seq data with three sub-workflow options for preprocessing that leverage 10x Genomics Cell Ranger ATAC software, the ultra-fast Chromap procedures, and a set of custom scripts implementing current best practices for scATAC-seq data preprocessing. The pipeline extends the R package ArchR for downstream analysis with added support to any eukaryotic species with an annotated reference genome. Importantly, scATACpipe generates an all-in-one HTML report for the entire analysis and outputs cluster-specific BAM, BED, and BigWig files for visualization in a genome browser. scATACpipe eliminates the need for users to chain different tools together and facilitates reproducible and comprehensive analyses of scATAC-seq data from raw reads to various biological insights with minimal changes of configuration settings for different computing environments or species. By applying it to public datasets, we illustrated the utility, flexibility, versatility, and reliability of our pipeline, and demonstrated that our scATACpipe outperforms other workflows.

Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.

De novo sphingolipid biosynthesis necessitates detoxification in cancer cells.

Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.

Repression of essential cell cycle genes increases cellular fitness.

A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells.