LSF small molecule inhibitors phenocopy LSF-targeted siRNAs causing mitotic defects and senescence in cancer cells

The oncogene LSF has been proposed as a novel target with therapeutic potential for multiple cancers. LSF overexpression correlates with poor prognosis for both liver and colorectal cancers, for which there are currently limited therapeutic treatment options. In particular, molecularly targeted therapies for hepatocellular carcinoma targeting cellular receptors and kinases have yielded disappointing clinical results, providing an urgency for targeting distinct mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models of hepatocellular carcinoma, with no observable toxicity. To understand how the inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Phenotypically, inhibition of LSF activity induced a mitotic delay with condensed, but unaligned, chromosomes. This mitotic disruption resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. The cellular phenotypes observed upon FQI1 treatment were due specifically to the loss of LSF activity, as siRNA specifically targeting LSF produced nearly identical phenotypes. Taken together, these findings confirm that LSF is a promising therapeutic target for cancer treatment. Significance Specific inhibition of LSF by either small molecules or siRNA results in mitotic defects resulting in cell death or senescence, supporting the promise for LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs. hepatocellular carcinoma cell lines.


Introduction
LSF (encoded by TFCP2) is an evolutionarily conserved transcription factor that is normally expressed ubiquitously at low levels, but is significantly overexpressed in hepatocellular carcinoma cell lines and patient samples. Levels of LSF in patient samples from multiple populations rise with increased stage and severity of disease (1)(2)(3)(4). Furthermore, LSF is oncogenic for hepatocellular carcinoma, as it is sufficient, in the background of a nontumorigenic, but tumor-primed hepatocyte cell line, for hepatocellular carcinoma tumor growth in mouse xenograft models (1). Elevated LSF levels have also been documented in a number of other cancers, as well (5). In both colorectal cancer and hepatocellular carcinoma, patients with elevated LSF levels have significantly worse prognosis, with shorter median disease-free survival times than those with low LSF levels (3,6). Finally, recent reports demonstrated that LSF can function as a co-activator for key transcription factors downstream of the Hippo and Wnt signaling pathways -YAP (4) and β -catenin (7) -both of which are widely accepted to contribute to liver proliferation and oncogenesis, as well as other cancer types.
Primary liver cancer and colorectal cancer are among the most common cancers worldwide (sixth and third, respectively), and represent leading causes of cancer mortality (second and fourth, respectively) (8)(9)(10). Although treatment options have improved, patients are often diagnosed with late stage, metastatic disease, resulting in high mortality. Hepatocellular carcinoma represents approximately 70-80% of primary liver cancer cases (9,11). The only two FDA-approved therapies for late-stage hepatocellular carcinoma, Sorafenib and Regorafenib (multi-kinase inhibitors), unfortunately demonstrate only modest improvement in patient survival rates (12,13), and result in significant side effects and rapid development of drug resistance.
Thus, a large unmet medical need remains for hepatocellular carcinoma and colorectal patient populations. Therapies directed to distinct molecular targets, ideally to which the cancer is oncogene addicted, have been promoted for mitigating these diseases (11).
A family of small molecule inhibitors of LSF, Factor Quinolinone Inhibitors (FQIs), was identified that inhibits the DNA binding and transcription activity of LSF, but not that of transcription factors from multiple other structural classes (14). Activity of p53, the closest structural relative of the LSF family (15,16), was also not inhibited by FQIs. Phenotypically, FQIs inhibit growth of hepatocellular carcinoma cells in vitro. They also inhibit hepatocellular . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint carcinoma tumor growth in vivo in multiple mouse models, including a mouse endogenous liver tumor model (17). In all cases, inhibition of tumor growth occurred in the absence of toxicity, as assessed by liver injury markers, histopathology of tissues with rapid cell turnover, or blood cell counts (18). These results suggested that hepatocellular carcinoma cells are oncogene addicted to LSF (14,19).
Oncogenic transcription factors are promising therapeutic targets given that they regulate tumorigenic pathways. However, transcription factors, in general, have been notoriously difficult to target with small molecule inhibitors as their DNA binding domains are commonly small and the proteins themselves are intrinsically disordered promiscuity (20). Identification of the transcription factor LSF as an oncogene and the significant inhibition of tumor growth upon LSF inhibition with no observed toxicity indicate that LSF holds considerable promise as a cancer therapeutic target (1,14,21). Targeting a transcription factor has been challenging, therefore validation of the biological specificity of the LSF inhibitors is essential. Here we demonstrate that the molecular and phenotypic consequences of knockdown of LSF with a specific siRNA are the same as treatment of cells with FQI1, therefore confirming that FQIs are highly specific in targeting this transcription factor. The molecular mechanisms by which LSF promotes cancer cell survival has not been well characterized, although initial data indicated that FQIs induce a "prometaphase-like" arrest in hepatocellular carcinoma cells (17). Clarifying the pathways by which inhibition of LSF leads to cell death is important to further support the candidacy of FQIs as a molecular therapy. Cell cycle analysis by flow cytometry and time-lapse microscopy revealed mitotic defects including mitotic delays with condensed, but unaligned chromosomes, leading to increased time in mitosis, defective cell division, multi-nucleation, and apoptosis. In addition, loss of LSF activity induced senescence in a sub-population of cells in a dose-dependent manner. Senescence, as well as mitotic arrest and apoptosis, are all desirable outcomes for a cancer chemotherapeutic.
The siRNA control was a sequence targeting RNA encoding firefly luciferase and was, therefore, non-targeting in the cells utilized for these studies. Cells were transfected using RNAimax (Life Technologies) according to manufacturer's instructions. Transfection efficiency was measured by fluorescent microscopy 24 hours post transfection by cellular uptake of the Cy3 labeled control siRNA, and was determined to be >90%. For all siRNA experiments, the initial thymidine block was started 24 hours after transfection of the siRNA.

Time-lapse microscopy
In order to image cell cycle progression for HeLa cells,   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint Infrared detection quantitated each band on an individual pixel basis using western analysis tools in the Image Studio program.

Gene expression determination
For most experiments, RNA was isolated using the Qiagen RNAeasy kit following the manufacturer's instructions. cDNA was generated using a Reverse Transcription kit from

Data Availability
The ChIP-seq data have been submitted to GEO. The data that support the findings of this study are available from the corresponding author upon request.

Chemical inhibition of LSF induces mitotic defects
Previously, we reported that LSF inhibition by small molecule inhibitors (FQIs) resulted in cells delayed with G2/M ("4n") DNA content in hepatocellular carcinoma cell lines.
. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint Furthermore, fluorescent staining of synchronized cells for DNA and tubulin demonstrated that when control cells completed mitosis and re-entered G1, FQI-treated cells generally remained in mitosis with condensed but unaligned DNA (17). In order to elucidate the basis for these FQI1mediated mitotic defects and their consequences in greater detail, and over a range of FQI1 concentrations, we synchronized HeLa cells, which demonstrate the same phenotype, with a double thymidine block (Fig. 1A), and analyzed samples for cellular DNA content throughout the subsequent cell cycle. At 1.8 µM, FQI1-treated cells were initially delayed in returning from G2/M to G1, remaining with 4n DNA content, compared to control cells that had re-entered G1 In some cell lines, LSF is necessary for upregulation of thymidylate synthase expression and therefore efficient transition through S phase (25). However, our previous studies indicated that proper progression through mitosis.

LSF small molecule inhibition during cell synchronization results in reduced expression of key mitotic regulators
Upon LSF activity inhibition, we observed defects in chromosome alignment and segregation, resulting in mitotic delay and multi-nucleation: phenotypes previously reported following inhibition or deficiency of major mitotic regulators Aurora kinase B (AURKB) and Cyclin Division Cycle 20 (CDC20) (28,29). To investigate whether inhibition of LSF alters expression of AURKB and CDC20 RNA, HeLa cells were synchronized with a double thymidine block ( Fig. 2A). RNA levels were measured in vehicle-treated cells at the G1/S border (0 hours) and as cells progressed to or just through mitosis (8 hours transcript reduction, AURKB and CDC20 protein levels were also reduced in a dose-dependent manner ( Fig. 2C and 2D), whereas LSF protein levels were unchanged, as expected (Fig. 2C).
The impact of the downregulation of AURKB was tested by monitoring phosphorylation of an AURKB substrate. Phosphorylation of Histone 3 on Serine 10 (30) was reduced by FQI1 in a dose-dependent manner ( Fig. 2C and 2D).
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint 1 0 The mitotic phenotypes would indeed be consistent with inhibition of Aurora kinase B and/or CDC20. Aurora kinase B inhibition leads to defects in kinetochore-microtubule attachment and cytokinesis, followed by multinucleation (31,32), and knockdown of CDC20 results in an increase in mitotic time (29). In addition, both LSF and AURKB are upregulated in hepatocellular carcinoma patient samples, with each positively correlating with disease severity (1,33). However, in contrast to previous results in asynchronous cells (17), Cyclin B levels were also downregulated (Fig. 2C), particularly when cells were treated with the highest concentrations of FQI1 during the synchronization procedure. This finding suggested that cells treated with FQI1 during synchronization are not efficiently proceeding through the cell cycle into mitosis after the G1/S arrest, complicating straightforward interpretation of the gene expression results. Due to the induction of cell cycle defects upon LSF inhibition, the protein expression data could not therefore distinguish whether downregulation of AURKB and CDC20 was the cause or consequence of the phenotypic outcomes.
In order to distinguish whether diminished AURKB and CDC20 gene expression resulted from lack of cell cycle progression of LSF inhibited cells or from diminished expression of these genes in mitosis, we analyzed RNA in synchronized, LSF-inhibited cells only from cells in mitosis, isolated by the standard mitotic shakeoff methodology ( Supplementary Fig. S2A-B). A reproducible decrease in CDC20, but not AURKB, RNA was observed. As an alternative method to identify candidate LSF target genes, we sought to identify genes around which LSF binds. The absence of a sufficiently robust antibody against LSF for chromatin immunoprecipitation (ChIP) prevented testing for endogenous LSF. Instead, a HEK cell line expressing an inducible, HAtagged LSF (14) was used for initial ChIP-sequencing analysis. Multiple HA-LSF binding peaks were observed around the AURKB gene ( Supplementary Fig. S2C), and binding of LSF was validated both at the AURKB promoter and around 3000 bp upstream of the transcription start site by quantitative PCR (Supplementary Fig. S2D). In contrast, no HA-LSF binding peaks were observed within 20 kb of the CDC20 gene. Taken in combination, whether LSF activates AURKB expression in these, or other, cells remains unresolved. However, these data suggest that LSF may regulate CDC20 expression, although either indirectly or from distant binding sites.
Global gene expression data from cells treated with FQI1 between G1/S and mitosis did not . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint 1 1 identify dysregulation of any other mitotic regulators that would cause an early mitotic delay (34).

RNAi mediated knockdown of LSF phenocopies inhibition of LSF with the small molecule inhibitor FQI1
A critical aspect of interpreting the data with FQI1 was to determine whether its effects truly reflected inhibition of the identified target. ). In addition, since certain siRNAs can cause nonspecific reduction in mRNA encoding MAD2 (37), which controls the spindle assembly checkpoint, we verified that the selected siRNA targeting LSF did not inadvertently reduce MAD2L1 transcript levels ( Supplementary Fig. S3D).
In order to compare downstream molecular outcomes from FQI1 and LSF siRNA treatments, whether direct or indirect, AURKB or CDC20 RNA levels were measured following RNAi mediated knockdown of either LSF or a non-expressed control. HeLa cells were transfected with siRNAs, to initiate protein knockdown, 30 hours prior to synchronization. RNA and protein expression were analyzed at two time points -when control cells were arrested at G1/S (0 hours) and when these cells were largely in mitosis after release from the block (8 hours) (Fig. 3A, Supplementary Fig. S4A). For ease of comparison, RNA levels were plotted relative to the level in the control siRNA sample at each time point. At 20 nM siRNA, significant knockdown of both LSF-encoding RNA (Fig. 3B) and protein (Fig. 3C) were achieved over this time course. Consistent with the results generated with the LSF small molecule inhibitor at 8 hours after G1/S release (Fig. 2B), AURKB and CDC20 RNA levels were reduced (Fig. 3B).
Immunoblotting of lysates harvested at the approximate time of mitotic entry of the control cells (8 hours) confirmed a dose-dependent reduction in AURKB and CDC20 protein levels after . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint siRNA-mediated knockdown of LSF ( Fig. 3C and D), consistent with the findings upon inhibition of LSF with FQI1 (Fig. 2C). As expected, phosphorylation of AURKB substrates Serine 10 and 28 of histone H3 (30, 38) was reduced. As with FQI1 treatments, Cyclin B levels were also reduced in a dose-dependent manner. Thus, LSF siRNA phenocopied the molecular consequences of FQI1 on gene expression, whether due to direct transcriptional effects, and/or consequences of cell cycle dysregulation.
To determine whether LSF knockdown resulted in similar cellular phenotypes to those observed with FQI1, synchronized YFP-H2B-expressing HeLa cells were transfected with siRNAs targeting LSF or a non-expressed control. A single thymidine block protocol was sufficient for synchronization ( Fig. 4A), as mitotic progression is viewed on a cell-by-cell basis.
Representative time-lapse images of cells treated with the highest concentration (20 nM) of either LSF targeting siRNA or control siRNA highlight dramatic changes in mitotic progression.
Control cells exhibited progression through normal mitotic phases in a timely manner (Fig. 4B).
However, cells with diminished LSF levels exhibited an extensive delay with condensed, but unaligned chromosomes, generally followed by defective cellular division and multinucleation (Fig. 4B). In addition, some cells remained in mitosis with condensed chromosomes throughout the entire time lapse analysis. Upon quantitation mitotic time was dramatically increased when LSF levels were reduced (Fig. 4C). Counterintuitively, the lower concentrations of LSF siRNA resulted in longer times for mitotic progression. We propose that at the higher LSF knockdown conditions the more defective cells are unlikely to enter mitosis, thus selecting for a less severe population available for the analysis. Indeed, when siRNA-transfected cells were imaged by time lapse microscopy after a double thymidine block, there was an inverse correlation between higher levels of LSF knockdown and the number of cells capable of entering mitosis ( Supplementary Fig. S4B).
By histone H2B fluorescence, the most striking mitotic outcome for individual cells treated with LSF siRNA after the extended delay in mitosis appeared to be mitotic slippage (Fig. 4B).
This was confirmed by immunofluorescence of synchronized cell populations, co-stained for DNA and α -tubulin ( Supplementary Fig. S5A-B). Quantitation of these immunofluorescence data demonstrated significant increases in cells with condensed, but nonaligned chromosomes, incomplete cytokinesis, and multinucleation upon LSF knockdown. These phenotypes mimicked . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint those observed with FQI1 treatment (Supplementary Fig. S5C-D). Finally, both types of treatments yielded mitotic cells with cellular protrusions (Supplementary Fig. S5E).
Also consistent with the results from FQI1 treatment, knockdown of LSF did not induce phosphorylated H2AX (γ-H2AX) foci, as monitored the beginning of mitosis in a synchronized cell population (Fig. 4E). This result suggests that the effects of inhibiting LSF on mitotic progression are not due to defects induced indirectly in S phase. Since

Induction of cellular senescence following inhibition of LSF
In addition to mitotic delay resulting from LSF inhibition, some cells undergoing synchronization while inhibiting LSF were arrested at other points in the cell cycle:cellular DNA profiling of FQI1-or LSF siRNA-treated cells being synchronized with a double thymidine block, captured cells that no longer progressed from the 2n state into S phase upon release from the G1/S block (Fig. 1B, Supplementary Fig. S4A), and time-lapse microscopy showed that a considerable fraction of the cells treated with LSF siRNA during a double thymidine block never entered mitosis during 10-12 hours after release from the G1/S block (Supplementary Fig. S4B).
Mitotic defects, caused by multiple distinct insults, can lead to senescence after G1 re-entry with  (Fig. 5A-B). Overall, there was a 3-to 5-fold increase in senescent cells with increasing amounts of LSF inhibition, although treatment with 0.9 μ M FQI1 was not sufficient to induce senescence. These data show that inhibition of LSF can result in senescence of cancer cells, and support the hypothesis that reduced LSF levels or activity during previous cell cycle(s) can predispose cells to senescence.

Discussion
The transcription factor LSF is an oncogene in multiple cancer types, notably including hepatocellular carcinoma (1,5,21). Small molecule inhibitors directly targeting LSF inhibited hepatocellular carcinoma cell proliferation in vitro and tumor growth in vivo with no signs of toxicity at doses required for tumor inhibition (14, 17,18). Together, these data indicate that LSF is a promising therapeutic candidate for hepatocellular carcinoma patients, and likely for other cancer types. The robust anti-tumor activity of FQIs is consistent with the initial report that a dominant negative LSF reduced tumorigenicity (1). Given the difficulties generally encountered in targeting transcription factors with small molecules (20), further investigation was warranted to confirm that anti-tumor effects of FQIs were due to specific targeting of LSF. Here, we demonstrated that a siRNA targeting LSF produced strikingly similar results to that of FQI1 treatment in all aspects, confirming specific targeting by the small molecule inhibitor.
Furthermore, knockdown of the close LSF paralog, LBP1A, did not result in such mitotic defects. Thus, we conclude that LSF is the factor required for accurate and efficient mitotic progression in these cancer cells.
The simplest interpretation from the mitotic phenotypes observed is that, as a transcription factor, LSF directly regulates expression of mitotic regulators. Indeed, both FQI1 and LSF siRNA result in downregulation of AURKB and CDC20 expression. However, further . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint examination indicates that these consequences may be indirect. Furthermore, it is noteworthy that unlike the emphasis on Cyclin B levels in a previous report (17) in which asynchronous cells were treated with FQI1, when synchronized cells were examined Cyclin B protein levels in 1.8 µM FQI1-treated cells were similar to those in control cells, and certainly not increased (Fig.   2C). We conclude that the increase in cyclin B protein levels observed in Rajasekaran et al. It has been previously demonstrated that LSF inhibition limits tumor progression, and that LSF inhibition causes apoptosis in multiple hepatocellular carcinoma cell lines (14, 17), as demonstrated here by HeLa cells with sub-G1 cellular DNA content (Fig. 1A, Supplementary   Fig. S4). Here, we report for the first time that senescence can also be induced by LSF inhibition in cancer cells. Using both small molecule inhibition and siRNA knockdown, the percentage of senescent cells was proportional to the extent of inhibition of LSF during the synchronization protocol (Fig. 5). Both senescence and apoptosis are desirable outcomes for treatment of cancer.
Since both small molecule inhibitors and siRNAs targeting LSF can lead to cancer cell death or senescence in vitro, it is worthwhile to consider the targeting strategy for LSF inhibition in patients. Many cancer drug candidates target mitosis in an effort to exploit this key vulnerability of cancer cells. However, many such therapies have failed in trials, which may result from: (1) tumor escape, where pathway redundancy or evasive resistance in mammalian cells enables the tumor cell to escape the therapy (43,44), or (2) low mitotic index where the drug half-life may not be long enough to suppress the target when cell division is triggered for any particular tumor cell (45,46). As a target, LSF may have an advantage toward avoiding tumor escape.
Inhibiting a transcription factor can target multiple pathways simultaneously, thus the likelihood that system redundancy would fully compensate is diminished. In addition, the issue of low mitotic index may be avoidable for the LSF inhibitors, since the apparent lack of toxicity in preclinical models may permit dosing in manners that generate sustained drug levels. Gene The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint silencing based approaches may also provide a useful strategy to counter low mitotic index for hepatocellular carcinoma patients. The first RNAi drug, which uses a lipid nanoparticle to encapsulate and efficiently deliver siRNA to hepatocytes, was recently approved following robust and durable gene silencing over the 18-month pivotal study (47). Additionally, a ligandbased strategy to deliver LSF siRNA to hepatocytes may provide added benefit as recent human data using a triantennary N-acetylgalactosamine (GalNAc) mediated siRNA delivery system demonstrated robust knockdown of a hepatic target that was sustained for more than a year (48,49). The target of GalNAc, asialoglycoprotein receptor (ASGR1) (23), is expressed , in early stages and often in later stages of hepatocellular carcinoma (50), although whether tumors retain ubiquitous expression is not clear.
In summary, comparing cellular and molecular outcomes of small molecule inhibitors that eliminate LSF activity to those of RNAi that specifically targets LSF, the specificity of FQI1 for LSF was confirmed. Both mechanisms resulted in similar mitotic defects, followed by cellular death or senescence, proving that LSF regulates mitosis in cancer cells. Therefore, the anti-tumor activity of FQI1 in multiple preclinical models is explicitly due to loss of LSF activity. These findings support the candidacy of LSF targeting agents for treatment of hepatocellular carcinoma and other cancers in which LSF is identified as an oncogene.      The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/665570 doi: bioRxiv preprint 2 8

Figure 5
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