Analysis of replication-coupled histones

Abstract

Methods for predicting cancer aggressiveness and / or risk of cancer recurrence in a subject are provided. The methods can include the steps of detecting histone gene expression at one or more replication-coupled histone genes in a biological sample from the subject and comparing the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of cancer aggressiveness and / or risk of cancer recurrence. Also disclosed are methods of ameliorating cancer aggressiveness and / or risk of cancer recurrence in a subject by reducing elevated histone gene expression at one or more replication-coupled histone genes in a subject.

Description

Attorney Docket No.1426.48.WO ANALYSIS OF REPLICATION-COUPLED HISTONES STATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 683,342, filed on August 15, 2024, the entire contents of which are incorporated by reference herein. STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING A Sequence Listing in XML format, entitled 1426-48WO_ST26.xml, 41,158 bytes in size, generated on August 14, 2025, and filed herewith, is hereby incorporated by reference into the specification for its disclosures. FIELD OF INVENTION

[0002] The invention relates to assays for predicting cancer aggressiveness and / or risk of cancer recurrence in a subject. The methods can include the steps of detecting histone gene expression at one or more replication-coupled histone genes in a biological sample. The invention also relates to methods of ameliorating cancer aggressiveness and / or risk of cancer recurrence which can include steps of reducing elevated histone gene expression at one or more replication- coupled histone genes in a subject. BACKGROUND

[0003] Hypertranscription is common in stem / progenitor cells (Kim, Y.K., et al., Absolute scaling of single-cell transcriptomes identifies pervasive hypertranscription in adult stem and progenitor cells. Cell Rep, 2023.42(1): p.111978), and in human cancers hypertranscription predicts poor prognosis (Zatzman, M., et al., Widespread hypertranscription in aggressive human cancers. Sci Adv, 2022.8(47): p. eabn0238.). For example, misregulation of the MYC transcription factor has been observed in most human cancers (Y. Dong, R. Tu, H. Liu, G. Qing, Regulation of cancer cell metabolism: oncogenic MYC in the driver's seat. Signal transduction and targeted therapy 5, 124 (2020)), though exactly how increased MYC binding to gene regulatory elements drives cancer has been controversial. However, it is difficult to reconcile promiscuous incremental increases in expression of thousands of genes with the presumed direct action of oncogenic transcription factors in activating expression of target genes to drive tissue-Attorney Docket No.1426.48.WO specific malignancies. Additionally, detection of hypertranscription relies on mRNAs, which are heavily processed and have variable half-lives, and on accurate cell number estimations.

[0004] Alternatively, hypertranscription in cancer may be relevant only to the subset of genes producing protein products that are rate-limiting for proliferation. For example, the multi-subunit enzyme, ribonucleotide reductase (RNR) – which is required for converting RNA bases to DNA bases – is rate-limiting for DNA synthesis, and RNR activity is a target of widely used anti- cancer catalytic inhibitors (S. E. Huff, J. M. Winter, C. G. Dealwis, Inhibitors of the Cancer Target Ribonucleotide Reductase, Past and Present. Biomolecules 12, (2022)). Similarly, chromatin components such as histones are rate-limiting for proliferation (F. Lu et al., Integrator- mediated clustering of poised RNA polymerase II synchronizes histone transcription. bioRxiv DOI: 10.1101 / 2023.10.07.561364, (2024); S. K. Huang, P. H. Whitney, S. Dutta, S. Y. Shvartsman, C. A. Rushlow, Spatial organization of transcribing loci during early genome activation in Drosophila. Curr. Biol.31, 5102 (2021)), insofar as all newly synthesized DNA must be packaged into nucleosomes every cell cycle, and the 64 genes encoding all 5 histone subunits must produce ~5% of a human cell’s total protein during S-phase (R. Milo, R. Phillips, Cell Biology by the numbers. (Garland, 2016)). Replication-coupled (RC) histone mRNAs are not polyadenylated and so are essentially absent from RNA-seq libraries, and the possibility that they drive over-proliferation has been overlooked.

[0005] Accordingly, despite the advances in the art, there remains a need for facile and accurate analyses to identify aggressiveness of a cancer and risk of recurrence in cancer samples to inform treatment in a subject. It would be an advance in the art to provide an affordable and rapid assay of a variety of biological samples and cancers for clinical applications and retrospective studies. This disclosure addresses these and related needs. SUMMARY

[0006] Embodiments of the present invention are based, in part, on the development of assays for hypertranscription of the histone genes that are exclusively transcribed during S-phase that can be utilized for identifying risk of cancer aggressiveness and risk for recurrence.

[0007] In an aspect, methods of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject are provided, comprising detecting histone gene expression at one or more replication-coupled histone genes in a biological sample from the subject and comparingAttorney Docket No.1426.48.WO the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of cancer aggressiveness and / or risk of cancer recurrence.

[0008] In another aspect, methods of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject are provided, comprising detecting chromatin accessibility at one or more replication-coupled histone genes in a biological sample from the subject and comparing the chromatin accessibility at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of chromatin accessibility at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of cancer aggressiveness and / or risk of cancer recurrence.

[0009] In another aspect, methods of monitoring cancer progression in a subject are provided, the methods comprising detecting histone gene expression at one or more replication-coupled histone genes on samples obtained at two more points in time from the same subject; and comparing the level of histone gene expression in each sample to a reference and / or to each other, wherein an increase in the level of histone gene expression in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression in the subject. The method may further comprise administering a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation to the subject with cancer progression.

[0010] In an additional aspect, methods of monitoring cancer progression in a subject are provided, the method comprising detecting chromatin accessibility at one or more replication- coupled histone genes on samples obtained at two more points in time from the same subject; comparing the level of histone gene expression in each sample to a reference and / or to each other, wherein an increase in the level of chromatin accessibility in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression in the subject. The method may further comprise administering a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation to the subject with cancer progression.

[0011] In a further aspect, the disclosure provides a kit comprising primers and / or probes specific to one or more replication-coupled histone genes and a reverse transcriptase.Attorney Docket No.1426.48.WO

[0012] These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS.1A-1S. RNA polymerase II (RNAPII) -Ser5p formalin-fixed paraffin-embedded (FFPE)- Cleavage Under Targeted Accessible Chromatin (CUTAC) directly maps hypertranscription. (1A) Model for hypertranscription in cancer: Paused RNA Polymerase II (RNAPII) at active gene regulatory elements such as promoters and enhancers increases on average over the cell cycle, resulting in a net proportional gain in RNAPII occupancy across the genome. Using RNAPII FFPE-CUTAC we can map hypertranscription genome-wide using three complementary approaches: 1) Genome-scaled Tumor (T) minus Normal (N) counts at candidate cis-regulatory elements (cCREs), 2) T – N at S-phase-dependent histone genes and 3) SEACR Tumor peak calls using Normal as the background control. (1B-1E) T – N versus log(T + N) / 2 plots showing hypertranscription mapped over the 343,731 annotated mouse cCREs for tumor and normal sections dissected post-tagmentation from a 10 micron FFPE slice from each of four different paraffin blocks. Hypertranscription of a cCRE is defined as the excess of RNAPII- Ser5p in the indicated tumor over normal (Tumor minus Normal in normalized count units for Mm10-mapped fragments pooled from the same slide). (1F-1M) All fragments were pooled from four slides from the same paraffin block and the number of fragments equalized between tumor and normal for each of the seven cancers. T – N versus log(T + N) / 2 plots showing hypertranscription mapped over the 984,834 annotated mouse cCREs for tumor and matched normal sections from 5 micron FFPE slices. Max Diffs displays the Tumor minus Normal maximum of the seven samples for each cCRE. (1N-1S) For each of the indicated tumors, tracks are shown for 50-kb regions around the #1-ranked cCRE based on Tumor and Normal counts. Raw data tracks were group-autoscaled together for tumor and normal, where SEACR Tumor peak calls (light gray) use Normal as the negative control. Gene annotations and cCREs (black rectangles) are shown at the top.

[0014] FIGS.2A-2J. RNAPII levels identify likely HER2 amplifications and regions of linkage disequilibrium. (2A-2B) Normalized count tracks and SEACR peak calls for the 1-Mb region on Chromosome 17q21 (2A) and for the 250-kb 17q12 region (2B) amplified in the breast tumor but evidently not in the colon tumor. Tracks were group-autoscaled together for Tumor (dark gray)Attorney Docket No.1426.48.WO and Normal (gray), where SEACR Tumor peak calls (light gray) use Normal as the negative control. Broad regions of prominent RNAPII hypertranscription indicate likely HER2 amplifications in both tumors. (2C-2D) Bins of 1-kb were tiled over each 1 Mb region centered on the highest peak in Chr17q21 (2C) corresponding to the ERBB2 promoter in Chr17q21, and the RFFL promoter in Chr17q12 (2D), and count density within each bin is plotted with curve- fitting and smoothing. Each of the six summits in the Breast tumor sample is centered over the promoter peak indicated by an arrow. (2E-2H) Same as (2C-2D) for top-ranked loci outside of the HER2 amplicon. (2I) Individual broad summits in (2D-2E) were zoomed-in and rescaled on the x-axis centered over the indicated promoter peak and superimposed over data tracks scaled to the height of the central peak. (2J) Data tracks for the CCNK promoter region, where the normalized count increase in the Breast tumor relative to normal over the 10-kb region shown is 5.4-fold and for Colon is 2.1-fold. The range for the other five tumors is 0.9-2.5.

[0015] FIGS.3A-3I. RNAPII over histone genes correlates with aggressiveness in meningiomas (3A-3F) and breast tumors (3G-3I). (3A) UMAP of 114 human tumor samples and replicates including those from 30 meningiomas. HCC: Hepatocellular carcinoma; ICC: Intrahepatic cholangiocarcinoma. (3B) Same as (3A) colored for sequencing depth and indicating homogeneous tumor clusters. (3C) UMAP of RNAPII FFPE-CUTAC data based on 500-bp bins over the human genome. (3D) Same as (3C) for cCREs. (3E) Same as (3C) for the 64 S-phase- dependent histone genes. (3F) WHO grade correlates best with RNAPII occupancy over histone genes. (3G) Same as (3A) excluding meningiomas but including samples and replicates from 13 additional breast tumors. (3H) Same as (3E) for breast tumor samples and replicates, colored for tumor type. (3I) Same as (3H) colored for tumor percentage.

[0016] FIGS.4A-4B. RNAPII over histone genes uniquely predicts recurrence in meningiomas. (4A) UMAPs of FFPE-CUTAC and frozen RNA-seq meningioma patient samples grouped by different biomarkers. From the left to right: RNAPII over S-phase-dependent histone genes; RNAPII over ribosomal protein genes; Percent of Chromosome M (chrM = Mitochondrial DNA); Chr22q loss. UMAPs are based on integrating FFPE-CUTAC (circles) with frozen-RNA- seq (triangles) samples (FIG.15) using the canonical correlation analysis. For RC histone and ribosomal protein genes, points are colored by high (dark gray) or low (gray for histone genes and darker gray for ribosomal protein genes) RNAPII enrichment over the corresponding genes for FFPE-CUTAC samples or their shared nearest neighbor RNA-seq samples. For chrM, pointsAttorney Docket No.1426.48.WO are colored by low (gray) or high (dark grey) fractions of Chromosome M fragments, with the hypothesis that a low chrM fraction predicts malignant. For chr22q loss, the FFPE-CUTAC samples with chr22q loss and their RNA-seq samples are colored dark gray, while the ones with chr22q intact are colored gray. (4B) Kaplan-Meier (KM) plots of recurrence-free rate as a function of time in months based on each grouping strategy using biomarkers. For fair comparisons between Histone genes, Ribosomal Protein genes and chrM, the same number of samples, i.e., the top five patients, are selected in the malignant group. The fraction of the chrM predictor has settings (6 to 18 samples in the malignant group) with small p values. However, the KM curve order is in the wrong direction for the hypothesis that a low fraction of chrM predicts fast recurrence.

[0017] FIGS.5A-5D. RNAPII over histone genes predicts whole-arm chromosome losses. (5A) P-values from the log-rank test for the KM separation of malignant from benign for all thresholds of the 30 meningioma patients and the corresponding 36 FFPE-CUTAC samples for Histone genes, Ribosomal protein genes, and % mitochondrial DNA (FIGS.4A-4B), also including the combined aneuploidy data for all 39 chromosome arms. (5B) Scatterplots and Spearman correlations between RNAPII over histone genes and number of whole-arm gains (upper panel) and losses (lower panel) for each of the 30 meningioma patient samples. (5C) Spearman correlations between the signal for RNAPII over histone genes and null RNAPII occupancies at cCREs for all 39 autosomal arms for Meningiomas (upper panel) and Breast tumors (lower panel). This summarizes correlation coefficients and significances on the correlation scatterplot in FIG.19. A negative correlation is expected if gains are more frequent than losses (i.e., chromosome arm gain will lead to a lower percentage of cCREs regions with zero RNAPII signals) with increasing RC histone gene signal and a positive correlation if losses are more frequent than gains. The error bar describing the standard deviation of the Spearman correlation coefficient is based on 1000 bootstrap estimation. The significance level is indicated by the stars on the top of each bar with *: p-value < 0.05, **: p-value <0.01, and ***: p-value < 0.001. (5D) Model for induction of centromere breaks and aneuploidy by histone gene hypertranscription. Histone H3|H4 dimers (spheres) are deposited along chromosome arms during DNA replication for chromatin duplication, while CENPA|H4 dimers (red-green spheres) are deposited at centromeres and maintain centromere identity. H3|H4 and CENPA|H4 histone dimers are preferentially deposited (arrows) at the CAF1 and HJURP assembly factors,Attorney Docket No.1426.48.WO respectively. However, the excess H3|H4 dimers produced by hypertranscription in cancer cells compete with CENPA|H4 for centromere assembly, reducing centromere function and creating DNA breaks (Example 1, References 38, 39). Segregation of broken chromosomes leads to whole-arm aneuploidies.

[0018] FIGS.6A-6H. RNAPII-Ser5p FFPE-CUTAC shows stronger and more frequent changes in up-regulation than down-regulation of cCREs. The Voom / Limma option of the Degust server (degust.erc.monash.edu / ) was applied to mouse cCRE RNAPII-Ser5p FFPE-CUTAC data from pooled replicates from 5 RELA and 4 PDGFB experiments. MA plots display x = log10(Tumor*Normal) / 2 versus y = log2(Tumor / Normal) for normalized counts from the tumor and normal samples being compared, and red color indicates FDR < 0.05. Normalized counts are the fraction of counts at each base pair scaled by the size of the Mm10 reference sequence (2,818,974,548), so that if the counts are uniformly distributed across the reference sequence, there would be one at each position. (6A-6B) Both RELA and PDGFB tumor sections show higher counts than normal sections but significant RELA changes both up and down are far stronger than PDGFB changes, confirmed in a head-to-head comparison between tumors and normal sections. (6C-6E) Same as (6A-6B) except using either RNAPII-Ser5p or histone H3K27ac antibodies for FFPE-CUTAC and using entire 10-μm curls divided into 4-8 samples per curl for PCR and sequencing. For MA plots, data were merged from multiple experiments and equalized by downsampling to 10 million fragments, with 4 merged replicates per sample. DAP-stained slides for each paraffin block used, with the total fraction of tumor indicated in parentheses. (6F-6H) Voom / Limma was used to construct MA plots based on individual 10-μm sections from single slides corresponding to the boxed sections on slides DAP-stained for tumor- driver transgene expression. Numbers in parentheses are percentages of tumor cells based on the numbers of stained and unstained cells within the boxed sections. Relative to normal, more cCREs increase in RNAPII than decrease.

[0019] FIG.7. Hypertranscription mapped over the 343,731 ENCODE-annotated mouse cCREs categorized by regulatory element type. For each tumor and normal sample, we counted the number of mapped fragments spanning each base-pair in a cCRE scaled to the mouse genome and averaged the number of counts over that cCRE across tumor or normal samples. We then divided up the 343,731 cCREs into the five ENCODE-annotated categories: Promoters (24,114), H3K4me3-marked cCREs (10,538), Proximal Enhancers (108,474), Distal Enhancers (211,185)Attorney Docket No.1426.48.WO and CTCF cCREs (24,072) and rank-ordered based on tumor minus normal representing global upregulation. Conversely, we rank-ordered cCREs based on normal minus tumor representing global downregulation. With such a large collection of loci, our a priori expectation is that the rank-ordered distribution of differences between tumor and normal will be approximately the same regardless of whether the differences are based on tumor minus normal or normal minus tumor. For clarity, we plotted rank-ordered differences on a log10scale. Strong hypertranscription for RELA and PDGFB-1, weak hypertranscription for PDGFB-2, and little or no hypotranscription for YAP1, are seen for all classes, consistent with the T – N versus log10(T + N) / 2 plots shown in FIGS.1B-1E.

[0020] FIG.8. Photographs of 5-μm FFPE sections from human tumor and adjacent normal tissues. Pathology classification, age and sex were provided by the vendor (BioChain). Each image spans the width of a standard charged microscope slide, where the tissue is visible under the paraffin skin. On-slide RNAPII-Ser5p FFPE-CUTAC was applied to slides in parallel, using a total of four slides each for 100 separate samples in all to produce the data analyzed in this study.

[0021] FIGS.9A-9X. Hypertranscription in human Tumor-vs-Normal tissues. Related to FIG. 2. (9A-9H) Same data as in FIGS.2A-H, except plotted as in FIG.7 to facilitate comparisons. (9I-9P) Combined data from a single slide with duplicate removal. (9Q-9X) Combined data from 4 slides after removing duplicates and equalizing the number of fragments between tumor and normal sections. The number of unique fragments per sample in each Tumor / Normal pair is Breast: 1,125,608; Colon: 3,712,097; Kidney: 2,031,893; Liver: 2,983,411; Lung: 1,123,638; Rectum: 3,284,736; Stomach: 719,598.

[0022] FIGS.10A-10D. Genome browser tracks for the top-ranked CRE regions for Breast (10A), Colon (10B), Lung (10C), and Stomach (10D). For each of the indicated tumors, tracks are shown for 50-kb regions around the #1-ranked cCRE based on Tumor and Normal counts. Data tracks were group-autoscaled together for Tumor and Normal, where SEACR Tumor peak calls (light gray) use Normal as the negative control. Gene annotations and cCREs (black rectangles) are shown at the top. The #1-ranked cCREs intersected promoters in the Breast, Colon and Lung samples and intersected an intergenic enhancer in the HSP90AA1 gene in the Stomach sample.Attorney Docket No.1426.48.WO

[0023] FIGS.11A-11F. Hypertranscription differs between human liver tumors. (11A-11D) Top-ranked cCREs based on liver tumors 1 and 2 and matched normal counts. Tumor / Normal tracks and Tumors 3-5 are group-autoscaled. (11E) Same as (11A), except for the minor histone gene cluster on Chromosome 1. (11F) Levels of hypertranscription differ between different hepatocarcinomas (Tumor 1: solid lines, Tumor 2 dotted lines). For each tumor and normal sample, we counted the number of mapped fragments spanning each base-pair in a cCRE scaled to the human genome and averaged the number of counts over that cCRE. We rank-ordered based on tumor minus normal representing global upregulation, and conversely rank-ordered cCREs based on normal minus tumor representing global downregulation. With such a large collection of loci, our a priori expectation is that the rank-ordered distribution of differences between tumor and normal will be approximately the same regardless of whether the differences are based on tumor minus normal or normal minus tumor. For clarity, we plotted rank-ordered differences on a log10scale.

[0024] FIGS.12A-12D. FFPE-CUTAC mitochondrial DNA signal is reduced in tumors. (12A) The percentage of normalized counts mapping to Chromosome M (chrM = mitochondrial DNA) was calculated for FFPE-CUTAC data from four mouse brain tumor paraffin blocks driven by PDGFB, YAP1 and RELA transgenes. An RNAPII-Ser5p antibody was used for the first four comparisons, and an RNAPII-Ser2p and histone H3K27ac antibodies were used respectively for the fifth and sixth comparisons. (12B) Same as (12A) for RNAPII-Ser5p FFPE-CUTAC data for the seven human Tumor / Normal pairs used in this study. (12C-12D) ATAC-seq count data from TCGA (tumor) and ENCODE (normal) shows variability in chrM percentages between tumors, consistent with our finding based on FFPE-CUTAC.

[0025] FIGS.13A-13L. Focal hypertranscribed regulatory elements embedded in broad regions of hypertranscription on Chromosome 17q12-22. (13A-13F) The six most highly transcribed cCREs within the ~5 Mb region of Chromosome 17q1.2-2.2 are displayed with each tumor and normal pair scaled to one another so that peaks can be observed in all samples. SEACR peaks are group-autoscaled in all panels. (13G-13L) Same as (13A-13F) except that all tumor-normal samples are group-autoscaled to the height of the tallest peak, where the disappearance of all the peaks except for those in Breast and for MSL1 and ERBB2 in Colon is evidence that peaks in these regions are strongly hypertranscribed in Breast and partially in Colon but not in any of the other tumors.Attorney Docket No.1426.48.WO

[0026] FIGS.14A-14H. HER2 amplifications account for most of the hypertranscription signal in the Breast and Colon cancer samples. FIGS.14A-14H correspond to FIGS.1F-1M, where the superimposed red dots are cCREs within Chr17q12-21.

[0027] FIGS.15A-15C. SEACR identifies and precisely maps amplifications in cancer. SEACR peaks were called on a merged set of RNAPII-Ser5p CUTAC datasets from K562 cells, which are annotated for amplifications in human genome build hg19. An amplified region in K562 cells on Chr22q is shown as a UCSC browser track at (15A) 25 Mb and (15B) at 600 kb scales, together with SEACR broad peaks called on published K562 RNAPII-Ser5p CUTAC datasets (19, 20). Dotted line indicates the location of the BCR-ABL t(9;22)(q34;q11) translocation breakpoint on Chr22q. (15C) The precise correspondence to the annotated amplified regions is evident in the SEACR tracks and in 9 autoscaled tracks from 3 separate experiments, where the breakpoint in each gene (marked by the dotted line) precisely corresponds to the change in signal amplitude, with abrupt increases within the part of each gene that is amplified, precisely mapped by SEACR.

[0028] FIGS.16A-16B. Hypertranscription at S-phase dependent histone genes. (16A) IGV tracks of the major histone gene cluster on Chromosome 13 from fusion-transgene-driven mouse brain tumors. Tumor and Normal 10-μm sections were from the same slide. Slides used for PDGFB-2a-c were from the same paraffin block but used in different experiments, and all others were from different paraffin blocks. Numbers at right were obtained by subtracting the sum of normalized counts in the normal sections from that in the tumor sections over all 64 annotated single-exon S-phase-dependent histone genes, where the Standard Deviation is shown. Paired t- test: * p < 0.001; ** p < 0.00001. (16B) IGV tracks of the human minor histone gene cluster on Chromosome 1 where tracks are autoscaled for each Tumor and Normal. Tumor and Normal 5- μm sections were from a matched pair of slides taken from the same patient.

[0029] FIG.17. Integration of FFPE-CUTAC samples with frozen RNA-seq meningioma. Green circles mark frozen RNA-seq samples (triangles) with a close and matched FFPE-CUTAC neighbor (dots), and red circles mark RNA-seq samples without a close matching FFPE-CUTAC neighbor. Points are colored by the WHO grade.

[0030] FIGS.18A-18F. RNAPII at 64 histone genes predicts recurrence time of meningiomas but RNAPII at Ribosomal Protein genes or fraction of mitochondrial DNA do not. (16A) Density distribution of the RNAPII at Histone genes (left) and Ribosomal Protein genes (middle), and theAttorney Docket No.1426.48.WO fraction of chrM (left) across 30 meningioma patients and 36 FFPE-CUTAC samples. The hypothesis is that high RNAPII signals or a low fraction of chrM predict faster recurrence. Threshold 4.4 (grey line) in the Histone gene panel is set to separate the malignant from the benign group used in FIGS.4C-4D, considering most meningioma patients are benign. Dashed lines correspond to a series of separation thresholds to illustrate the survival changing pattern in 16B-16D. (18B-18D) Kaplan-Meier (KM) curves compare the recurrence between the malignant and benign group predicted by RNAPII signals at 64 PC Histone genes (18B) and Ribosomal Protein genes (18C) and the fraction of chrM (18D). The sample number in each panel title indicates the sample number in the predicted malignant group. The p values at the left-bottom corner of each panel are the log-rank test evaluating the separation of two survival curves.95% confidence intervals are shown as ribbons around the curves. The dashed square highlights the significant thresholding settings for chrM, but the KM curve order is against the hypothesis that low chrM predicts malignancy. (18E-18F) Evaluation of the recurrence difference between patients with any chromosome 1q (chr1q) and chromosome 6p (chr6p) gain (18E) or any loss (18F), where histone genes major cluster is located on chr1q and the minor cluster is located on chr6p.

[0031] FIGS.19A-19B. Whole-arm losses are more frequent than whole-arm gains in meningiomas as RNAPII signals increase on histone genes. To design a metric to quantify the relative frequencies of gains and losses for each of the 39 human autosomal arms (1p, 1q,...22q), we measured the percentage of cCRE regions with zero counts spanned by all cCREs on that arm for all meningiomas (19A) and breast tumors (19B). This yielded a single aneuploidy indicator value for each patient, whereby if the chromosome arm is lost, we should observe closer to 100% of cCRE regions with zero counts on that arm. We plotted this aneuploidy indicator on the y-axis against the aggregated RNAPII counts over the RC histone genes on the x-axis for that patient tumor, where the p-value for Spearman correlation is shown at the bottom of each panel. FIG.5 summarizes the correlation coefficients and significance levels on each arm. Excess of whole- arm losses (-1 / 2 dose) versus whole-arm gains (+3 / 2 dose) is counter-intuitive, as 1 / 500 newborns carry a trisomy but no newborn with a monosomy is known to have come to term.

[0032] FIG.20. Overview of an example general PCR test for cancer aggressiveness.Attorney Docket No.1426.48.WO

[0033] FIGS.21A-21B. Exemplary nested PCR primers for histone gene RNA:DNA PCRs (21A) Example primer design for Histone H2B (21B) Example nested primers for replication- coupled histones.

[0034] FIGS.22A-22C. Whole-arm losses correlate with recurrence in meningioma and across 33 TCGA cancer types. (22A) Whole chromosome arm gains and losses were identified using CaSpER across 1,298 meningioma RNA-seq samples. Whole-arm aneuploidy is defined as the total number of chromosome arm gains or losses. Kaplan-Meier curves compare recurrence times among patients with low (fewer than three), medium (three to six), and high (more than six) levels of chromosome arm alterations. The log-rank test p-values, shown in the lower-left corner of each panel, assess the statistical significance of survival curve separation. (22B) Chromosome arm aneuploidy was determined using the ABSOLUTE algorithm in 10,522 TCGA whole-genome sequencing (WGS) samples, as reported in Example 3, Ref. [8], Table S2. Disease-free time serves as the recurrence metric. (22C) Relationship between median disease- free time and the average number of whole chromosome arm losses (left) or gains (right) across 33 TCGA cancer types. The solid lines represent linear regression fits. The kidney chromophobe (KICH) cancer type has a significantly longer disease-free time, exceeding the range displayed in the figure.

[0035] FIG.23. Percentage of whole-arm gains and losses in selected TCGA data. Each histogram bar represents the overall percentage of intact, gained and lost chromosome arms in TCGA data for the indicated cancer type (Table 3), where ACC is adrenocortical carcinoma, BRCA is breast cancer, LAML is acute myeloid leukemia and GBM is glioblastoma. For clarity, only 4 of the 33 cancer types are shown. The full TCGA set of histograms is displayed in FIG. 27. Vertical black lines separate whole chromosomes, where Chromosomes 13, 14, 15, 21 and 22 are acrocentrics with a short p arm (not displayed) and long q arm.

[0036] FIGS.24A-24E. Acrocentrics and metacentrics gain or lose whole arms at similar frequencies in cancer. (24A) A metacentric segregation; (24B) An acrocentric segregation; (24C) A metacentric segregation following a centromere break; (24D) An acrocentric segregation following a centromere break. Acrocentric p chromosome arms are short and lack unique loci; thus, intact acrocentrics are not distinguishable from q arms resulting from centromere breaks in genomic studies. (24E) TCGA whole-genome sequencing (WGS) data by tumor type. Each dot represents a different autosomal chromosome arm (5 acrocentric long arms and 17 metacentrics).Attorney Docket No.1426.48.WO

[0037] FIGS.25A-25B. Model for generation of centromere breaks leading to losses > gains. (25A) Cancer progression: Normal cells (grey) proliferate and differentiate to populate tissues, but aberrant induction of histone overexpression (black) promotes both hyperplasia and chromosome instability. Selection in hyperproliferating clones drives the frequencies of certain chromosomal abnormalities and the evolution of malignant cellular features. (25B) Model: Defective centromeres compromised by centromeric histone displacement will break, leading to widespread aneuploidy through whole chromosome arm loss, arm gain, and through whole chromosome loss. The occurrence of micronuclei by encapsulation of fragmented chromosome arms further stimulates chromosomal instability.

[0038] FIG.26. S-phase-dependent histone mRNAs are under-represented in meningioma patient RNA-seq data. Log-scale hexbin plot compares the gene-by-gene distribution of average RNAPII fragment counts from FFPE-CUTAC to average RNA-seq transcript counts from frozen meningioma samples of the same patients. The individual histone gene signals are indicated as dark circles, where the large majority are high in RNAPII but very low for RNA-seq. Exceptions in the upper right quadrant are likely to represent S-phase-independent replacement histones, including histone partners for H2A.X, H2A.Z, H3.3 and CENP-A variant histones, which are encoded by intron-containing genes outside of the S-phase-dependent histone clusters.

[0039] FIG.27. Percentage of whole-arm gains and losses in TCGA data. See the description for FIG.23.

[0040] FIGS.28A-28C. Acrocentric and metacentric whole-arm aneuploidies are recovered at similar frequencies in 33 cancer types. Whole-genome sequencing data was used from 10,674 cancer patients spanning 33 cancer types downloaded from The Cancer Genome Atlas (TCGA, portal.gdc.cancer.gov). For 32 of 33 cancer types, no significant differences are seen between acrocentrics and metacentrics in the frequencies of whole-arm gains, losses or both gains and losses (Wilcoxon rank test), except for OV, in which metacentrics are in excess at p < 0.05. Each dot represents a different autosomal chromosome arm (5 acrocentric long arms and 17 metacentrics). The chromosome arm gain or loss was inferred using ABSOLUTE

[0038] profiles for each patient. The major and minor alleles of each segment were summed and the minimum and maximum of the copy number taken across segments on each chromosome arm. Any increases in the minimal allelic copy number from the diploid copy number of 2 indicate an arm gain. Similarly, any decreases in the maximum allelic copy number from the diploid copyAttorney Docket No.1426.48.WO number of 2 indicate a whole-arm loss for the corresponding autosomal arm. (28A) Gains; (28B) Losses; (28C) Gains or losses.

[0041] FIG.29. Acrocentric and metacentric whole-arm aneuploidies are recovered at similar frequencies in Nanopore long-read RNA-seq data. See the description for FIG.24. Data are from combined B-ALL and AML leukemia patient samples described in Ref (18) of Example 3.

[0042] FIGS.30A-30B. Chromatin factors and RNAPII at the histone locus in wing imaginal disc cells. (30A) Browser tracks of chromatin factors and RNAPII isoforms across chromosome 2L of Drosophila. The arrowhead marks the location of the histone locus. (30B) Browser tracks of chromatin factors and RNAPII isoforms across one Histone Repeat Unit.

[0043] FIGS.31A-31B. Histone modifications at the histone locus in wing imaginal disc cells. (31A) Per-gene copy abundance of chromatin features and histone modifications in wildtype cells (log2 CPM), and the fold-change in per-gene copy abundance between the 12XWT strain (12 histone gene copies) and wildtype (100 copies). (31B) Browser tracks of histone modifications around the histone locus in wildtype wing imaginal disc cells. Shading marks the histone locus.

[0044] FIGS.32A-32B. Expression of a His2ADendra2 reporter Histone Repeat Unit in wildtype and 12XWT strains. (32A) Fluorescence of the His2ADendra2 reporter in a wildtype larval wing imaginal disc, adjusted to display the weak fluorescence expressed from the reporter in this genotype. (32B) Fluorescence of His2ADendra2 in larval wing imaginal discs from wildtype (wt) and from 12XWT larvae. Fluorescence in 12XWT background is 37X that in the wildtype background (average summed fluorescence of wildtype discs 5,908,005 + 1,096,058 a.u. versus 158,704 + 26,029 a.u. in the 12XWT background).

[0045] FIGS.33A-33M. Silencing and derepression of extra histone genes in the Drosophila male germline. (33A) The HLB in testis cells is marked by Mxc staining. Cells in the proliferating zone at the apical tip of the testis are marked with phospho-Mxc detected by the MPM2 antibody at the HLB (gray). Post-mitotic G2 phase germline cells are marked by bamGAL4-induced UASRFP expression (dark gray). The proliferating zone of the testes is marked by the light gray bar, and the RFP-marked G2 gonial cells by the dark gray bar. (33B- 33H) Fluorescence of histone-Dendra2 reporter constructs (gray) in live testes. The apical tip is marked with an asterisk, the proliferating zone is outlined with white dashed lines on phase contrast images, and G2 phase gonial cells are identified with RFP. (33B, 33C) His2AVDendra2Attorney Docket No.1426.48.WO expression in wildtype males. Dendra2 fluorescence is apparent throughout the testis, including in the apical tip, and intensely stains the nuclei of isolated and squashed gonial cells, marked with bamGAL4-induced RFP expression. (33D, 33E) His2ADendra2 Histone Repeat Unit expression in wildtype males. Fluorescence is absent from the proliferating zone and from RFP- positive gonial cells but is present in later germline cells. (33F, 33G) His3Dendra2 Histone Repeat Unit expression in wildtype males. Staining is absent throughout the germline; the few fluorescent nuclei are in somatic cells of the testis sheath. (33H) Fluorescence of the His2ADendra2 HRU reporter in 12XWT males. This line does not carry bamGAL4 and UAS- RFP constructs; the proliferating zone was identified by position in the testis (dashed line). Strong H2ADendra2 fluorescence is apparent throughout the apical tip of the testis and in later stages. (33I) Results of tests for derepression of His2ADendra2 and His3Dendra2 Histone Repeat Unit reporters in testes with reductions in chromatin factors. Su(var)3-9 was tested in Su(var)3-91 / Su(var)3-92homozygotes, all other factors were tested by bamGAL4-induced knockdown in testes. Dark squares represent repression similar to wildtype, light squares indicate derepression. (33J-33M) Derepression of His2ADendra2 (33J, 33K, light gray) or of His3Dendra2 (33L, 33M, light gray) in cells with bamGAL4-induced RFP expression (dark gray) and His4 knockdown (KD). Fluorescence is absent in the apical tip of the testis but appears in the post-mitotic stage where knockdown occurs.

[0046] FIGS.34A-34I. Histone H4 is a component of the Histone Locus Body in male germline cells. (34A-34C) Fresh squashes of testes with bamGAL4-induced expression of GFP-tagged histones (gray) Induced RFP expression (dark gray) marks G2 phase gonial cells. Histone H3- GFP (34A) and histone H3.3-GFP (34B) broadly label the nuclei of gonial cells. In contrast, induced histone H4-GFP (34C) labels one bright dot with a low broad background in G2 phase nuclei. (34D) The induced histone H4-GFP (light gray) dot coincides with the HLB, marked by Mxc staining (dark gray). (34E, 34F) Representative spermatogonial cells labeled with EdU (light gray) to mark nuclei with ongoing DNA replication or in gap phase.94% (47 / 50) S phase cells show focal staining of histone H4 (gray) and the bight white HLB dot is also visible in gap phase cells. (34G) Representative spermatogonial cell marked with the M phase epitope H3S10p (gray). The histone H4 dot is visible in only 7% (1 / 14) of mitotic cells. (34H) Kc167 nucleus stained with antibodies to histone H4 (dark gray) and Mxc (gray). Histone H4 is enriched in HLBs. (34I) Browser tracks of histone modifications around the histone locus in Kc167 cells.Attorney Docket No.1426.48.WO The histone locus is enriched for the active H3K27 acetylation modification, the RNAPII-S5p isoform, Mxc, and for histone H4.

[0047] FIGS.35A-35E. RNAPII isoforms at the HLB in the Drosophila testis. (35A) RNAPII- S5p (light gray) stains the HLB in the proliferating zone marked by phospho-Mxc staining (gray) and the HLB in more distal cells. Mxc staining (dark gray) marks the HLB. (35B) The proliferating zone is marked with phospho-Mxc (dark gray) and the G2 phase gonial cells by bamGAL4-induced RFP (gray). RNAPII-S5p staining (light gray) strongly stains the HLB in the proliferating zone, and more weakly stains the HLB in G2 phase cells. (35C) zoom of the boxed area in (35B). The arrowhead points to an HLB in proliferating zone, and the arrow to an HLB in a G2 phase cell. (35D) RNAPII-S2p staining (light gray) strongly stains nuclei in the proliferating zone and in RFP-labeled G2 phase cells, but is not enriched in HLBs. (35E) zoom of the boxed area in (35D). The arrowhead points to an HLB in proliferating zone, and the arrow to an HLB in a G2 phase cell.

[0048] FIGS.36A-36F. Histone H4 localizes with reduced RNAPII in HLBs. (36A) Immunostaining for RNAPII-S5p (dark gray) in the apical tip of a testis with bamGAL4-induced expression of histone H4-GFP (light gray). G2 phase gonial cells are marked by induced RFP expression (gray). The intense dot of H4-GFP coincides with focal RNAPII-S5p signal in G2 phase gonial cells. (36B) zoom of the box marked in (36A) showing costaining of HLBs with RNAPI-S5p and H4-GFP in RFP-positive G2 phase gonial cells. RNAPII staining in these cells is lower than that in more apical cells in the proliferating zone. (36C-36F) Testes with bamGAL4-induced RFP expression (gray) marking G2 gonial cells and stained for phospho-Mxc (dark gray) and the RNAPII-S5p isoform (light gray). Dashed lines demarcate the proliferative zone from G2 phase cells based on RFP expression. A wildtype testis (36C, 36D) with high phospho-Mxc in the proliferative zone and low staining in G2 phase cells. RNAPII-S5p signal in the proliferative zone is high at some HLBs and moderate at others, and moderate at HLBs in the RFP-marked cells. (36E, 36F) A testis with knockdown of His4 (KD). High phospho-Mxc signal is apparent in the proliferative zone and persists in the RFP-labeled gonial cells. The RNAPII- S5p signal is more intense at the HLB in gonial cells than in cells of the proliferative zone.

[0049] FIGS.37A-37D. Histone H4 localizes to the promoters of active histone genes in human K562 cells. (37A) Immunostaining human K562 cells for the HLB factor NPAT (light gray) and histone H4 (gray). (37B) Distribution of histone H3K27acetylation, RNAPII-Sp5, NPAT, andAttorney Docket No.1426.48.WO histone H4 across a portion of the HIST1 histone cluster on chromosome 6. Arrowheads mark three active canonical histone genes on one side of this cluster, arrowheads mark two inactive histone genes, and an arrowhead marks the promoter of an adjacent active non-histone gene. NPAT and histone H4 coincide only at active histone genes, and are absent from inactive histone genes and from active non-histone genes. (37C) Zoom showing coincidence of the H3K27 acetylation mark, RNAPII-S5p, NPAT, and histone H4 at the promoters of two active histone genes. (37D) Summary of summed signal for chromatin features at the 94 histone genes in the human genome. Variant histone genes are indicated with an asterisk. Localization of anti-histone H4 signal to the multiple HLBs in a K562 nucleus. Each histone isotype and variant is ordered by expression (RNAPIIS5p signal).

[0050] FIGS.38A-38C. Overview of example protocol for histone analysis.

[0051] FIG.39. Illustration of standard curve and data ensuring primers bind to and amplify target precisely and efficiently in example method of the disclosure.

[0052] FIG.40. Example evaluation of qPCR efficiency by primer and cell line where slope of regression line can be utilized to calculate the amplification efficiency.

[0053] FIG.41. Example melt curves analysis (left) and tapestation results (right) for quality control in example method.

[0054] FIG.42. Sequence verification from example method by Plasmidsaurus, providing full length reads, 3k-6k raw reads, and consensus sequence of most abundant molecular specifies in sample confirmed presence of desired target histones.

[0055] FIG.43. Relative abundance of targets in leukemia (upper) and melanoma (lower) versus normal cells from example method. n=3 biological replicates per group, mean + / - SEM shown; unpaired on-tailed t-test P<0.05(*), P<0.01(**), P<0.0001 (***), ns= not significant. DETAILED DESCRIPTION

[0056] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention toAttorney Docket No.1426.48.WO those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0058] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0059] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0060] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0061] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0062] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0063] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0064] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).Attorney Docket No.1426.48.WO

[0065] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0066] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0067] The term “consists essentially of” (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., remodeling activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0068] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0069] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized),Attorney Docket No.1426.48.WO synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base- pairing abilities or increased resistance to nucleases.

[0070] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded. The terms "nucleotide sequence" "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "recombinant nucleic acid," "oligonucleotide" and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid molecules and / or nucleotide sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.831 - 1.835 and the World Intellectual Property Organization (WIPO) Standard ST.26. A "5' region" as used herein can mean the region of a polynucleotide that is nearest the 5' end of the polynucleotide. Thus, for example, an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide. A "3' region" as used herein can mean the region of a polynucleotide that is nearest the 3' end of the polynucleotide. Thus, for example, an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.

[0071] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. Identity can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence AnalysisAttorney Docket No.1426.48.WO in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0072] As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence as compared to a reference polypeptide.

[0073] As used herein, the phrase “substantially identical,” or “substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, the substantial identity exists over a region of consecutive nucleotides of a nucleotide sequence of the invention that is about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, or more nucleotides in length, and any range therein, up to the full length of the sequence. In some embodiments, the nucleotide sequences can be substantially identical over at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides). In some embodiments, a substantially identical nucleotide or protein sequence performs substantially the same function as the nucleotide (or encoded protein sequence) to which it is substantially identical.

[0074] In some embodiments, the subject is “in need of” or “in need thereof” a method of the present invention, for example, the subject has findings typically associated with cancer (e.g., breast cancer, lung cancer, and the like).

[0075] As used herein, the term “therapeutically effective amount” refers to an amount of a treatment used in accordance with the methods disclosed herein that elicit a therapeuticallyAttorney Docket No.1426.48.WO useful response in a subject, for example, reduces progression of cancer, delayed onset of growth of cancer cells, reduced metastatic spread, or inhibited or reduced tumor burden. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0076] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0077] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0078] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0079] The term “contact” or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.

[0080] In some embodiments, the disclosure is directed to methods of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject, comprising detecting histone gene expression at one or more replication-coupled histone genes in a biological sample from the subject and comparing the levels of histone gene expression at the one or more replication- coupled histone genes in the biological sample to a reference, wherein an increase in the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of cancer aggressiveness and / or higher risk of cancer recurrence. In some embodiments, detecting histone gene expression comprises detecting RNA Polymerase II (RNAPII) levels at histone genes in samples to identify subjects with increased cancer aggressiveness or cancer at risk of recurrence. Cancer aggressiveness, as used herein, refers to the rate that cancer cells grow and / or spread, and / or their abnormal appearance and / or behavior (e.g., dedifferentiation). Cancer recurrence as used herein comprises cancerAttorney Docket No.1426.48.WO relapse or return in the same and / or different part of the body after treatment and / or remission. The disclosure is based, in part, on the development of methods for prediction of tumor aggressiveness, e.g., risk of recurrence, based on RNAPII occupancy at replication coupled histone genes. Based on the development disclosed herein, methods of accurately predicting tumor aggressiveness based on RNAPII occupancy at histone genes both establishes an unanticipated cancer driver paradigm and allows for new approaches to diagnostic methodologies.

[0081] In some embodiments, methods of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject are provided, comprising detecting aneuploids in a biological sample from the subject and comparing the aneuploids in the biological sample to a reference, wherein an increase in the levels of aneuploids in the biological sample compared to the reference is indicative of cancer aggressiveness and / or risk of cancer recurrence. In some embodiments, the method can be used in combination with or in conjunction with the other methods of predicting cancer aggressiveness and / or risk of cancer recurrence disclosed herein. In some embodiments, the aneuploids are whole arm aneuploids or whole chromosome aneuploids. In some embodiments, whole arm aneuploid loss, or decrease relative to a reference is indicative of cancer aggressiveness and / or risk or cancer recurrence. In some embodiments, an increased risk of recurrence is defined as a shorter disease-free time relative to a reference, which may be, for example, an average time of disease-free recurrence for a population of subjects having the same cancer which may be further narrowed by age, clinical or pathological stage, treatment modalities, etc.

[0082] In some embodiments, methods of predicting risk of recurrence and / or cancer aggressiveness in a subject are provided, comprising performing a chromatin accessibility assay (e.g., Cleavage Under Targeted Accessible Chromatin(CUTAC)) at histone genes in a biological sample from the subject, the method comprising permeabilizing the sample; contacting the sample with a first affinity reagent that specifically binds to a histone, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules; excising the tagged DNA segment associated with theAttorney Docket No.1426.48.WO histone involved in transcription regulation; and determining the nucleotide sequence of the excised tagged DNA segment, thereby mapping transcriptional activity on chromatin to quantify increases or decreases in RNAPII at the histone genes. Example CUTAC methods are described, for example, in International Patent Publication WO 2022 / 056309 and modified CUTAC that enables high-throughput FFPE tissue analysis in International Patent Application WO 2024 / 249846 , each of which are incorporated herein by reference in their entirety.

[0083] In some embodiments, methods disclosed herein can further comprise detecting RNAPII at active gene regulatory elements, e.g., enhancers, to further detect and / or characterize a sample for cancer, with high RNAPII signals indicative of risk for faster recurrence / higher risk of recurrence. In some embodiments, detecting RNAPII is performed across the genome. Because mammalian transcription initiates at many enhancers, identifying sites of RNAPII pausing, whether or not a stable RNA product is normally produced, is desirable, with CUTAC identifying active promoters and enhancers that produce enhancer RNAs. See, Kaikkonen MU, et al. (2013) Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol Cell 51:310-325 and International Patent Publication WO 2022 / 056309. In some embodiments, the method comprises detecting RNAPII at non-coding regions, including, for example, enhancers (e.g., proxy enhancer activation. See, e.g., de Langen P, Hammal F, Guéret E, Mouren JC, Spinelli L, Ballester B. Characterizing intergenic transcription at RNA polymerase II binding sites in normal and cancer tissues. Cell Genom.2023 Sep 29;3(10):100411; doi: 10.1016 / j.xgen.2023.100411 (showing intergenic transcription at RNAPII-bound regions is a novel per-cancer and pan-cancer biomarker and providing an atlas (see FIG.1, data S1) of intergenic transcription using RNAPII binding sites to connect genomic and transcriptomic data in normal tissues and cancer samples), incorporated herein by reference in its entirety. Hypertranscription, as used herein, refers to a global increase in nascent transcription and can be measured across the genome, mapping hypertranscription at regulatory elements across the genome. In an example embodiment, hypertranscription can be quantified, which can comprise normalizing count differences between tumor tissue sample and normal tissue sample from the same subject and / or same sample, with an example approach for quantification of hypertranscription.

[0084] In some embodiments, methods of monitoring cancer progression in a subject are provided, the method comprising detecting histone gene expression at one or more replication-Attorney Docket No.1426.48.WO coupled histone genes on biological samples obtained at two more points in time from the same subject; comparing the level of histone gene expression in each sample to a reference and / or to each other, wherein an increase in the level of histone gene expression in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression in the subject. The method can further comprise administering treatment (e.g., a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation) to the subject with cancer progression. In some embodiments, the treatment can be the onset of treatment in a subject that has not previously been treated or is no longer being treated or a change in ongoing treatment (e.g., to a more aggressive treatment). Methods of progression, as used herein, can comprise, for example, metastatic spread, increase in number or degree of metastases, recurrence of cancer, increase in growth of primary tumor, and / or remaining cancer cells at tumor site that were previously slow growing or quiescent increasing in proliferation. In some embodiments, progression comprises cancer recurrence and / or metastases. In some embodiments, treatment ameliorates (e.g., improves) cancer aggressiveness and / or risk of recurrence.

[0085] In some embodiments, a method of monitoring a disease or disorder is provided, the method comprising performing a method as described herein from biological samples obtained at two or more points in time from the same subject and comparing an amount and / or the genomic location of a targeted protein ( e.g., replication dependent histones, or associated proteins) on chromatin and / or the transcriptional activity on chromatin in each sample to a reference and / or to each other. In some embodiments, the amount of protein or transcription can be indicative of worsening (e.g., increased disease) or improving disease (lessening of the disease). In some embodiments, the number of cells with aneuploidy in a sample of cells can be indicative of worsening (e.g., increased disease) or improving disease (lessening of the disease). For example, an increase in aneuploidy relative to a reference control can be indicative of worsening disease, while a decrease in aneuploidy in a sample relative to a reference control can be indicative of improving disease. In some embodiments, the aneuploidy is whole arm loss. Accordingly, in some embodiments, an increased amount of whole arm loss in a biological sample relative to a reference is indicative of worsening disease.

[0086] In some embodiments, a reference control may be an aggregate of normal or healthy subjects, e.g., one or more subjects without the disease. Such reference controls can include aAttorney Docket No.1426.48.WO healthy population of a particular age, gender, race or other variable. In some embodiments, the reference control comprises comparing a diseased sample to a normal sample from the subject, for example, matched tumor and normal tissue. In an example embodiment, diseased tissue and normal tissue are derived from the same tissue sample, e.g., from the same section or different sections. A reference control can be use with any of the methods disclosed herein.

[0087] In some embodiments, a method of monitoring a disease or disorder comprises determining efficacy of a treatment. In some embodiments, the method comprising performing a method as described herein from samples obtained at two or more points in time from the same subject receiving the treatment (e.g., before and / or after treatment and / or at one or more times during treatment) and comparing an amount and / or the genomic location of a targeted protein on chromatin and / or the transcriptional activity on chromatin in each sample to a reference and / or to each other. In some embodiments, determining efficacy of a treatment comprises measuring the histone gene expression, RNAPII levels, and / or chromatin accessibility at one or more replication-coupled histone genes wherein an increase is indicative of worsening (e.g., increased disease) and a decrease is indicative of improving disease (lessening of the disease) as thereby indicative of efficacy of the treatment. In some embodiments, the differences in the amounts of the histone gene expression, RNAPII levels, and / or chromatin accessibility at one or more replication-coupled histone genes at the two or more points in time indicate efficacy of a treatment of the disease or disorder in the subject. In addition, the method can monitor disease progression and / or make treatment decisions for subjects based on changes in the histone gene expression or RNAPII levels or chromatin accessibility at one or more replication-coupled histone genes.

[0088] For example, as described in Roth et al., PRMT5 activity sustains histone production to maintain genome integrity; doi:10.1101 / 2025.07.03.663002, incorporated herein by reference, the authors confirm the work described herein that Histone H4 is a feedback repressor of histone gene synthesis and show that PRMT5 methylation of the H4R3 arginine residue is the epigenetic modulator of repression by H4 via its co-localization with NPAT, using both a catalytic inhibitor and a co-factor inhibitor. Roth et al. further show that decreased histone gene transcription is observed within 15 minutes after palbociclib administration whereas other effects of the drug are not seen for hours or days. The rapidity of PRMT5 inhibition on histone genes but not on any other genes shown in Roth et al. is an example of how one can use the methods of measuringAttorney Docket No.1426.48.WO histone gene expression disclosed herein to detect effectiveness of treatment, for example, anti- cancer treatment targeting PRMT5, allowing rapid evaluation of compounds that target basic transcription machinery.

[0089] In some embodiments, the reference control may be an aggregate of normal or healthy subjects, e.g., one or more subjects without the disease. Such reference controls can include a healthy population of a particular age, gender, race or other variable. The reference control can also comprise healthy tissue from the subject and / or the sample comprising diseased tissue (e.g., tumor). In some embodiments, the first sample is obtained from a subject prior to beginning of treatment. In some embodiments, the second sample is obtained during and / or after treatment.

[0090] In some embodiments, a method of prognosing cancer in a subject is provided, the method comprising performing a method as described herein on a sample from a subject, and prognosing the cancer in the subject based on changes in the histone gene expression, RNAPII levels, and / or chromatin accessibility at one or more replication-coupled histone genes relative to a reference. Thus, using the disclosed methods, a profile of an interaction, for example, RNAs associated with replication dependent histones to thereby identify hypertranscription and / or endogenous RNA interactions, can be generated allowing correlation with cancer. In some embodiments, an interaction profile for a particular cancer, or for a particular subject, subpopulation or population, can be generated using the methods described herein that can be used for diagnosis or prognosis of subjects with a similar profile. Accordingly, aspects of the disclosed methods relate to correlating histone gene expression, RNAPII levels, and / or chromatin accessibility at one or more replication-coupled histone genes with diagnosis or prognosis.

[0091] In some embodiments, the methods disclosed herein detect increased presence of RNAPII at one or more replication-coupled histone genes. Replication-coupled, or replication-dependent, histone genes encode messenger RNAs (mRNAs) which differ from all other cellular mRNAs: instead of being polyadenylated, these mRNAs end in a stem-loop structure and do not contain introns, where the only processing is cleavage of the nascent transcript to form the 3’ end of the histone mRNA. Replication-coupled histone genes are found at four discrete loci in mammals and are conserved across species and syntenic. Seal, R.L., Denny, P., Bruford, E.A. et al. A standardized nomenclature for mammalian histone genes. Epigenetics & Chromatin 15, 34 (2022), incorporated herein by reference, in particular at FIGS 1A-1C. In the human genome theAttorney Docket No.1426.48.WO replication-coupled histone genes are primarily found on chromosome 6 and chromosome 1. Replication-coupled histone genes may be alternatively referred to in different naming conventions, for example, HIST1H2AA, “histone cluster 1 H2A family member a”, H2AC1, or “H2A clustered histone 1.”

[0092] In some embodiments, the one or more replication-coupled histone genes comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 replication-coupled histone genes. In some embodiments, the one or more histone genes comprises HIST2H3D, HIST2H4B, HIST2H3A, HIST2H2AA4, HIST2H2BC, HIST2H2AA4, HIST2H3A, HIST2H4B, HIST2H2AC, HIST2H2AB, HIST1H2AA, HIST1H2BA, HIST1H2APS1, HIST1H1A, HIST1H3A, HIST1H4A, HIST1H4B, HIST1H3B, HIST1H2AB, HIST1H2BB, HIST1H3C, HIST1H1C, HIST1H4C, HIST1H1T, HIST1H2BC, HIST1H2AC, HIST1H1E, HIST1H2BD, HIST1H2BE, HIST1H4D, HIST1H2AD, HIST1H2BF, HIST1H4E, HIST1H2BG, HIST1H2AE, HIST1H3E, HIST1H1D, HIST1H4F, HIST1H4G, HIST1H3F, HIST1H2BH, HIST1H3G, HIST1H2BI, HIST1H4H, HIST1H2BJ, HIST1H2AG, HIST1H4I, HIST1H2AH, HIST1H2BL, HIST1H2AI, HIST1H3H, HIST1H2AJ, HIST1H2BM, HIST1H4J, HIST1H4K, HIST1H2AK, HIST1H2BN, HIST1H2AL, HIST1H1B, HIST1H3I, HIST1H4L, HIST1H3J, HIST1H2AM, HIST1H2BO, and any combination thereof.

[0093] In some embodiments, the one or more replication-coupled histone genes comprises one or more of the following gene Reference Sequences: NM_001123375, NM_001034077, NM_001005464, NM_001040874, NR_036461, NM_001040874 _ chr1_149822627, NM_001005464_ chr1_149824180, NM_001034077_ chr1_149832329, NM_003517, NM_175065, NM_170745, NM_170610, NR_045125, NM_005325, NM_003529, NM_003538, NM_003544, NM_003537, NM_003513, NM_021062, NM_003531, NM_005319, NM_003542, NM_005323, NM_003526, NM_003512, NM_005321, NM_021063, NM_003523, NM_003539, NM_021065, NM_003522, NM_003545, NM_003518, NM_021052, NM_003532, NM_005320, NM_003540, NM_003547, NM_021018, NM_003524, NM_003534, NM_003525, NM_003543, NM_021058, NM_021064, NM_003495, NM_080596, NM_003519, NM_003509, NM_003536, NM_021066, NM_003521, NM_021968, NM_003541, NM_003510, NM_003520, NM_003511,Attorney Docket No.1426.48.WO NM_005322, NM_003533, NM_003546, NM_003535, NM_003514, NM_003527, and any combination thereof.

[0094] In some embodiments, the methods of detecting replication-coupled histone gene expression at one or more histone genes comprises amplifying replication dependent histone genes. Preparation of the sample for detecting replication-coupled histone gene expression can comprise solubilizing total nucleic acids from a biological sample, for example, using a chaotropic buffer, and eluting nucleic acids, for example RNA and / or DNA. In embodiments, the method can comprise adding histone anti-sense primer to eluted RNA, reverse-transcribing RNA to generate cDNA, treating the sample with RNaseH, and / or adding a spike-in oligonucleotide prior to subjecting the sample to PCR or qPCR with histone-specific primers. In embodiments, the method can comprise eluting DNA, adding a spike-in oligonucleotide and subjecting the sample to PCR or qPCR. In some embodiments, the method comprises detecting amounts of cDNA and amounts of genomic DNA (gDNA). In some embodiments, the methods can comprise calculating a cDNA:gDNA ratio using spike-in oligonucleotide as an internal calibration standard.

[0095] In some embodiments, one or more probes or primers specific to one or more replication- coupled histone genes, e.g., a set, can be provided for use with the detection methods disclosed herein. In an embodiment, PCR primers are provided. In some embodiments, the PCR primers are designed to amplify all histone mRNAs. As described in the working examples, PCR primers can be designed to the most conserved regions identified of a Clustal-W multiple alignment of histone coding sequences. In some embodiments, the primers specific for a particular set of histone genes (e.g., histone H1, H2A, H2B, H3 or H4) comprise similar melting temperatures, for example, temperatures within about 15 °C of each other, e.g., within about 15 °C, 14 °C, 13 °C, 12 °C, 11 °C, or 10 °C. In some embodiments, the PCR primers are nested primers, with the primers having a length between 10 and 28 nucleotides, e.g., 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. The nested primers can be specific for replication coupled histones, for example, to amplify histone H1, H2A, H2B, H3, or H4. In some embodiments, nested primers can be provided to provide increased specificity for each histone. See, e.g., FIG.21B.

[0096] In some embodiments, spike-in oligonucleotide sequences can be used as a calibration standard, for example, an oligonucleotide with a length of about 30-75 nucleotides, for example,Attorney Docket No.1426.48.WO 40-60 nucleotides, 45-55 nucleotides, or any range therein. In some embodiments, the spike-in oligonucleotide sequence comprises primer-complementary sequences at either end of the sequence. In embodiments of the methods described herein, detection of histone gene hypertranscription can be calculated as a ratio of cDNA to genomic DNA using the cDNA:gDNA ratio of the spike-in oligonucleotide that is configured to be used as an internal calibration standard.

[0097] In some embodiments, detecting RNAPII levels comprises subjecting the sample to CUTAC with an affinity agent comprising an antibody to a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.

[0098] In some embodiments, a reference is used in the methods. In some embodiments, the reference is from non-cancerous tissue. The reference may be a threshold value, above which is indicative of hypertranscription. In an embodiment, the reference is based on a population of subjects, for example, a population of subjects of a particular age range, gender, and / or health status.

[0099] The sample may comprise tissue, blood, and / or cell-free DNA. In some embodiments, the sample is a biopsy sample. In some embodiments, the sample is from a tumor, e.g., a solid tumor. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample, a fresh tissue sample, or a frozen tissue sample. In some embodiments, the sample is from a subject with cancer. In some embodiments, the subject has brain, breast, colon, liver, lung, stomach, kidney or rectum cancer. In some embodiments, the brain cancer is a meningioma.

[0100] The methods as described herein can further comprise performing a chromatin assay and may include methods of preparing a library of excised chromatin DNA that is amenable to sequencing on any desired platform. In example embodiments, the chromatin assay is a chromatin accessibility assay. In some embodiments, methods can comprise chromatin profiling of samples. Chromatin profiling can include ATAC-seq, including, for example, scATAC, Fast- ATAC, an improved version of ATAC-seq that reduces mitochondrial reads (Corces MR et al. (2016) Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat Genet 48:1193-1203), and Omni-ATAC, an improved version that additionally improves signal-to-noise (Corces MR et al. (2017) An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14:959-962), andAttorney Docket No.1426.48.WO enzyme-tethering methods such as CUT&RUN (Skene, P., Henikoff, J. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13, 1006–1019 (2018)) and CUT&Tag (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019); Kaya-Okur, H.S., Janssens, D.H., Henikoff, J.G. et al. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264–3283 (2020)). In some embodiments, the chromatin profiling comprises chromatin accessibility, including for example, CUTAC (Henikoff S, Henikoff JG, Kaya-Okur, H.S., Ahmad, K, Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation. eLife 9:e63274 (2020)) and CUTAC in FFPE samples (Henikoff S, Henikoff JG, Ahmad K, Paranal RM, Janssens DH, Russell ZR, et al. Epigenomic analysis of Formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun. 2023;14:5930 (2024)).

[0101] In some embodiments, methods of predicting risk of recurrence and / or cancer aggressiveness can comprise a chromatin profiling assay, which may comprise analysis of RNAs associated with replication dependent histones using a reverse transcription and tagmentation assay (RT&Tag). In some embodiments, RT&Tag can be used to analyze RNAs associated with replication dependent histones and identify hypertranscription and / or endogenous RNA interactions. In some embodiments, RT&Tag can comprise steps of binding a first recognition agent that binds an epitope of the RNA associated with replication dependent histones, or its associated proteins, to a permeabilized nucleus, an organelle, a cell or a tissue that may be bound to a solid support; binding a second recognition agent that specifically binds to the first recognition agent, wherein the second recognition agent is conjugated to a biotin-binding moiety; tethering at least one molecule required for reverse transcription comprising a biotinylated oligonucleotide for cDNA synthesis priming and for PCR amplification to the second recognition agent; tethering a transposase fused to protein A (pA-transposase) comprising a first sequencing adapter sequence to the first recognition agent and the second recognition agent; allowing the at least one molecule required for reverse transcription to convert a mature transcript near the binding site of the first recognition agent to an RNA / DNA hybrid comprising a first sequencing adapter sequence and a priming sequence; allowing the pA-transposase to tagment the RNA / DNA hybrid; and preparing sequencing libraries of the RNA / DNA hybrid. See, Khyzha, N., Henikoff, S. & Ahmad, K. Profiling RNA at chromatin targets in situ by antibody-targetedAttorney Docket No.1426.48.WO tagmentation. Nat Methods 19, 1383–1392 (2022); International Patent Publication No. WO 2023 / 212580A1 each of which is incorporated by reference in their entirety.

[0102] The chromatin accessibility assay can include, but is not limited to, a Cleavage Under Targeted Accessible Chromatin (CUTAC) assay, an Assay for Transposase-Accessible Chromatin (ATAC)-seq assay, a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-seq assay (Bianco, S., Rodrigue, S., Murphy, B.D., Gévry, N. (2015). Global Mapping of Open Chromatin Regulatory Elements by Formaldehyde-Assisted Isolation of Regulatory Elements Followed by Sequencing (FAIRE-seq). In: Leblanc, B., Rodrigue, S. (eds) DNA- Protein Interactions. Methods in Molecular Biology, vol 1334), or a Nicking Enzyme-Assisted Accessible Chromatin Sequencing (NicE)-Seq assay (Ponnaluri, V.K.C., Zhang, G., Estève, PO. et al. NicE-seq: high resolution open chromatin profiling. Genome Biol 18, 122 (2017)). The method can comprise performing CUTAC wherein an affinity agent comprises an antibody to a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.

[0103] In some embodiments, the method is performed on a solid support, for example a bead, a slide, a well (e.g., a microwell or nanowell) and / or the wall of a microtiter plate. The bead may be an amine-functionalized bead, for example, an agarose-glutathione bead or a lectin-coated bead (e.g., Concanavalin A). In some embodiments, the bead is a magnetic bead. In some embodiments, the method is performed directly on a slide comprising the sample, e.g., a tissue sample. In example embodiments utilizing chromatin accessibility assays (e.g., CUTAC), the method can be performed on a slide to produce spatially resolved results, for example, as described in WO 2024 / 249846, which is incorporated herein by reference in its entirety. In some embodiments, the one or more probes or primers specific to one or more replication-coupled histone genes is provided in an array. An array may be provided on a solid surface, for example, a slide, microarray plate, or tubes.

[0104] In some embodiments, the subject has cancer and the method further comprises administering to the subject a prophylactic treatment, e.g., a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation to the subject identified at risk of cancer recurrence.

[0105] In some embodiments, the subject is identified as having a high risk of cancer aggressiveness, and the method further comprises administering a treatment course of a cancerAttorney Docket No.1426.48.WO biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation based on the high risk of cancer aggressiveness. As used herein, high risk of cancer aggressiveness

[0106] “Cancer” refers to an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize (spread). Specific cancers types include without limitation the cancers identified in Publication No. US 2006 / 0014949 and the following: cardiac: sarcoma (e.g., such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma and the like), myxoma, rhabdomyoma, fibroma, lipoma and teratomas; lung: bronchogenic carcinoma (e.g., such as squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma and the like), alveolar (e.g., such as bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal: esophagus (e.g., such as squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma and the like), stomach (e.g., such as carcinoma, lymphoma, leiomyosarcoma and the like), pancreas (e.g., such as ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma and the like), small bowel (e.g., such as adenocarcinoma, lymphoma, carcinoid tumors, Kaposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma, and the like), large bowel (e.g., such as adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma and the like); genitourinary tract: kidney (e.g., such as adenocarcinoma, Wilm’s tumor nephroblastoma, lymphoma, leukemia, and the like), bladder and urethra (e.g., such as squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma and the like), prostate (e.g., such as adenocarcinoma, sarcoma), testis (e.g., such as seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma and the like); liver: hepatoma (e.g., hepatocellular carcinoma and the like), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (e.g., such as osteosarcoma and the like), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma (e.g., such as reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (e.g., such as osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system: skull (e.g., such as osteoma, hemangioma, granuloma, xanthoma, osteitis deformans and the like), meninges (e.g., such as meningioma, meningiosarcoma, gliomatosis and the like), brain (e.g., such as astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma],Attorney Docket No.1426.48.WO glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors and the like), spinal cord (e.g., such as neurofibroma, meningioma, glioma, sarcoma and the like); gynecological: uterus (e.g., such as endometrial carcinoma and the like), cervix (e.g., such as cervical carcinoma, pre-tumor cervical dysplasia and the like), ovaries (e.g., such as ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma, and the like), vulva (e.g., such as squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma and the like), vagina (e.g., such as clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma) and the like); hematologic: blood (e.g., such as myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndromes and the like), Hodgkin’s disease, non-Hodgkin’s lymphoma; skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis and the like; and adrenal glands: neuroblastoma.

[0107] “Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. As used herein, the terms “treating” or "treatment" or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, reduce, slow down, lessen symptoms of, decrease and / or halt progression of cancer, e.g., cancer growth, metastases. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agentAttorney Docket No.1426.48.WO may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population) or as having a risk factor that increases the likelihood of developing the disease.

[0108] Examples of therapeutic agents include chemotherapeutics, biologically active agents, including immunotherapies, RNA therapeutics, biologics, and radiation therapies. Examples of a chemotherapeutic agent include without limitation: alkylating agents (e.g., which may include doxorubicin, cyclophosphamide, estramustine, carmustine, mitomycin, bleomycin and the like); antimetabolites (e.g., which may include 5-Fluoro-Uracil, capecitabine, gemcitabine, nelarabine, fludarabine, methotrexate and the like); platinating agents (e.g., which may include cisplatin, oxaliplatin, carboplatin and the like); topoisomerase inhibitors (e.g., which may include topotecan, irinotecan, etoposide and the like); tubulin agents (e.g., which may include paclitaxel, docetaxel, vinorelbine, vinblastine, vincristine, other taxanes, epothilones, and the like); signaling inhibitors (e.g., kinase inhibitors, antibodies, farnesyltransferase inhibitors, and the like); and other chemotherapeutic agents (e.g., tamoxifen, anti-mitotic agents such as polo-like kinase inhibitors or aurora kinase inhibitors, and the like).

[0109] Examples of radiation include without limitation, brachytherapy, photon beam radiation therapy (e.g., stereotactic radiosurgery, helical tomotherapy, three-dimensional conformal radiation therapy, cyberknife, gamma knife, fractionated radiosurgery), particle beam radiation therapy (proton beam radiation therapy) electron beam radiation therapy.

[0110] Immunotherapies can be used for treatments and can include chimeric antigen receptor (CAR) T-cell therapy, cancer therapy vaccines (e.g., sipuleucel-T, tamilogene laherparepvec), immunomodulators (e.g., thalidomide, lenalidomide, pomalidomide, imiquimod), monoclonal antibodies (e.g., antibody-drug conjugates, radiolabeled antibodies, bispecific T-cell engagers), immune checkpoint inhibitors (PD-1 and PD-L1 inhibitors, CLTA-4 inhibitors, LAG-3 inhibitors) and cytokines (e.g., chemokines such as interferons, tumor necrosis factors, and growth factors).Attorney Docket No.1426.48.WO

[0111] In some embodiments, methods of inhibiting expression of one or more replication- coupled histone mRNAs and / or mRNAs that translate into proteins functional at one or more replication-coupled histone genes are provided, and can comprise administering a therapeutic oligonucleotide, for example a therapeutic RNA, specific to a target mRNA (e.g., one or more replication-coupled histone mRNA and / or mRNAs that translate into proteins functional at one or more replication-coupled histone genes). In some embodiments, the target mRNA is transcribed from one or more replication-couple histone genes or one or more mRNAs translated into one or more protein components that function within the histone locus body or target the U7 small nuclear RNA. See, e.g., Marzluff and Koreski, Trends Genet.2017 Oct; 33(10): 745–759, incorporated herein by reference in its entirety.

[0112] In some embodiments, the replication-coupled histone gene or genes encodes histone H1, histone H2A, histone H2B, histone H3, or histone H4. In some embodiments, the replication- coupled histone gene or genes is as detailed elsewhere herein, including Table 2.

[0113] In some embodiments, the target mRNA translated into one or more protein components that function within the histone locus body or target the U7 small nuclear RNA is selected from nuclear protein at the ataxia-telangiectasia locus (NPAT), a stem-loop binding protein (SLBP), a U7 small nuclear ribonuclear protein, the human homolog of Drosophila melanogaster FLICE- associated huge protein (FLASH), and / or is a component of the histone cleavage complex (HCC). See, e.g., Geisler, M. S., et al. Histone locus bodies: a paradigm for how nuclear biomolecular condensates control cell cycle regulated gene expression. Nucleus, 14(1) (2023).

[0114] Example therapeutic RNAs include RNAi, such as siRNA, shRNA miRNA, antisense oligonucleotides, or decoy oligonucleotides. Example RNAi therapeutics can comprise a polynucleotide that is complementary to a portion of the target sequence mRNA of one or more replication-coupled histone genes, generally ranging in size from 15 to 50 base pairs. RNAi therapeutics include, for example shRNA and siRNA. In some embodiments, siRNA is a nucleic acid that can form a double stranded RNA with the ability to reduce or inhibit expression of a gene or target gene: each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length. A small hairpin RNA (shRNA) is also contemplated for use. The shRNA is an antisense strand of about 19 to about 25 nucleotides followed by a short nucleotide loop (approximately 5 to 9 nt) followed by the analogous sense strand. In an embodiment, an RNAi is a microRNA or miRNA,Attorney Docket No.1426.48.WO endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level, or synthetic versions of miRNAs. See, e.g., Lim et. al Science 299, 1540 (2003), Lee and Ambros Science, 294, 862 (2001), Lau et al., Science, 294, 858-861 (2001), Lagos-Quintana et al, RNA, 9, 175-179 (2003).

[0115] Different criteria are available for selecting the nucleic acid for use and may comprise scanning the mRNA sequence of the one or more replication-coupled histone mRNAs and / or mRNAs that translate into proteins functional at one or more replication-coupled histone genes, and may include empiric determination in accordance with, for example, Sui G et al., Proc. Natl. Acad. Sci. USA 99:5515-20 (2002), and may include confirmation the sequence lacks significant sequence homology with other genes as analyzed by BLAST search. Additional approaches may comprise any accessible site in endogenous mRNA that can be targeted for degradation by synthetic oligodeoxyribonucleotide / RNase H method (see, e.g., Lee N S et al., Nature Biotechnol.20:500-05 (2002)). RNAi treatment may comprise miRNA or siRNA, or a pre- miRNA which is processed by Dicer to form a miRNA. The RNAi may also comprise a dsRNA or shRNA which is processed by Dicer to form a siRNA. The polynucleotides may comprise one or more modifications to suppress innate immune activation, enhance activity and specificity, and reduce off-target induced toxicity. Example teachings can be found, for example at Provost et al., E.M.B.O. J., 2002 Nov.1; 21(21): 5864-5874; Tabara et al., Cell 2002, June 28; 109(7):861-71; Martinez et al., Cell 2002, September.6; 110(5):563; Hutvagner & Zamore, Science 2002, 297:2056. In certain embodiments, a single-stranded RNAi agent disclosed herein can comprise substitutions, or modifications, including chemically modified nucleotides, and non-nucleotides which may include incorporation in the backbone, sugars, bases, or nucleosides. The use of substituted or modified single-stranded RNAi agents can be designed to have an increased half-life in a subject. Furthermore, certain substitutions or modifications can be used to improve the bioavailability of single-stranded RNAi agents by targeting particular cells or tissues or improving cellular uptake of the single- stranded RNAi agents. Exemplary modifications and locations within a RNAi polynucleotide are described in Hu et al. “Therapeutic siRNA: state of the art” Signal Transduction and Targeted Therapy 5, Article number 100 (2020), incorporated herein by reference, see, e.g., Figures 2 and 3, specifically for its teachings of modifications.Attorney Docket No.1426.48.WO

[0116] In some embodiments, the therapeutic RNA is a decoy oligonucleotide that can be used according to methods disclosed herein. In some embodiments, the methods can comprise administering a decoy oligonucleotide specific to the target mRNA, e.g., one or more replication- coupled histone mRNA and / or mRNAs that translate into proteins functional at one or more replication-coupled histone genes. Shukla, T. N., Song, J., & Campbell, Z. T. (2020). Molecular entrapment by RNA: an emerging tool for disrupting protein–RNA interactions in vivo. RNA Biology, 17(4), 417–424.

[0117] In some embodiments, the subject has cancer, and the method further comprises the step of imaging the subject at a higher frequency for detection of cancer recurrence, cancer aggressiveness, and / or metastases relative to a subject without high risk of cancer aggressiveness and / or recurrence. In an example embodiment, increased surveillance can be utilized in a subject identified at higher risk, which may include, for example, increased cancer marker testing, increased testing of histone gene levels / RNAPII levels at histone genes and / or accessibility at histone genes according to the methods disclosed herein, and / or increased frequency of imaging by any modality. Example modalities include, but are not limited to, ultrasound, mammography, CT, PET-CT, MRI, and X-ray.

[0118] Compositions that can be used in the methods described herein are also provided. In some embodiments, a composition comprises primers and / or probes specific for a set of replication-couples histone genes. In some embodiments, a composition comprises a deparaffinized and permeabilized FFPE sample containing an RNAPII specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions. In some embodiments, a composition comprises a deparaffinized and permeabilized FFPE sample containing a chromatin protein specific affinity reagent that is linked directly or indirectly to a transposome in low ionic conditions.

[0119] In another aspect, the disclosure provides a kit of reagents, and optionally instructions, to facilitate performance of the methods described herein. In some embodiments, a kit of reagents can comprise reagents for the present assays and comprises two or more reagents including probes specific for a set of replication-coupled histone genes, reverse transcriptase, chaotropic buffers, and / or spike-in oligonucleotide calibration standards. In some embodiments, the kit comprises reagents for further analysis, for example FFPE CUTAC, two or more reagents (e.g., 3, 4, 5, or more) selected from a RNAPII-specific affinity reagent, one or more chromatinAttorney Docket No.1426.48.WO protein-specific affinity reagent, a SDS solution, a Triton®X-100 (octyl phenol ethoxylate) solution, a transposase solution, a tagmentation buffer, a cross-linking reversal solution, and amine-functionalized magnetic beads. These reagents are described in more detail above and all embodiments thereof are encompassed by this aspect and are not repeated here in detail. The kit may also comprise a low ionic solution to provide ionic conditions for transposase activity. The kit can optionally include written indicia (for example labels and / or instructions) directing the performance of the method as described herein. Such labeling and / or instructions can include, for example, information concerning the amount, and method of administration, detection and quantification for the assays detailed herein.

[0120] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention. EXAMPLES Example 1. RNA Polymerase II at histone genes predicts outcome in human cancer

[0121] Genome-wide hypertranscription is common in human cancer and predicts poor prognosis. To understand how hypertranscription might drive cancer, the FFPE-CUTAC method for mapping RNA polymerase II (RNAPII) genome-wide in formalin-fixed paraffin-embedded (FFPE) sections was applied. (Henikoff, S., et al., RNA Polymerase II hypertranscription in cancer FFPE samples. bioRxiv, 2024.). Global RNAPII elevations in mouse gliomas and assorted human tumors in small clinical samples were demonstrated and regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps discovered. RNAPII occupancy at S-phase-dependent histone genes correlated with WHO grade in meningiomas, accurately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneuploidy in cancer, with general implications for precision oncology.

[0122] Cleavage Under Targeted Accessible Chromatin (CUTAC) for Formalin-Fixed Paraffin- Embedded (FFPE) sections to directly map RNAPII in fixed tissue samples were adapted (6). ThisAttorney Docket No.1426.48.WO provides a DNA-based method for measuring transcription, instead of RNA-based methods that are limited by RNA instability and variable transcript half-lives. RNAPII was assessed across 343,731 candidate cis-regulatory elements (cCREs) defined by the ENCODE project (7) and compared normal mouse brain tissue to adjacent tumors induced by different transgene drivers (8- 10). It was observed that significantly upregulated cCREs were more frequent than downregulated cCREs (FIGS. 6A-6H). To sensitively detect RNAPII hypertranscription (FIG. 1A), where the absolute change is important, the number of mapped fragments was first counted spanning each base-pair in a cCRE scaled to the mouse genome coverage and averaged the normalized counts over that cCRE. Tumor minus Normal (T-N) counts on the y-axis was then plotted versus the average RNAPII signal (11) on a log10scale on the x-axis for clarity. This revealed clear hypertranscription (Tumor >> Normal RNAPII) for the RELA-driven tumor (FIG. 1B). Two PDGFB-driven tumors differed in hypertranscription, high in PDGFB-1 (FIG. 1C) and low in PDGFB-2 (FIG.1D), whereas the YAP1-driven tumor showed weak hypotranscription (FIG.1E). To determine whether hypertranscription is specific to any particular class of regulatory element(s), the data was divided into the five ENCODE-annotated cCRE categories and observed that the five RNAPII hypertranscription profiles are highly consistent with one another (FIG.7). This suggests that RNAPII abundance differences between tumors and normal brains affect all regulatory element classes.

[0123] The findings were next expanded to a diverse sample of human tumor and matched normal sections from the same patient (FIG.8). FFPE-CUTAC was performed and each pair rank-ordered by Tumor minus Normal (T-N) differences to test for RNAPII hypertranscription based on the 984,834 ENCODE-annotated human cCREs. Clear hypertranscription was observed in five of the seven tumors (breast, colon, liver, rectum and stomach) and for the composite of all samples (FIGS. 1F-1M, FIGS. 9A-9X). In contrast, the kidney and lung tumor samples tested showed essentially no hypertranscription, which implies that hypertranscription is a common, but not a defining feature of cancer (1, 12). A computational method independent of annotations using the SEACR (Sparse Enrichment Analysis for CUT&RUN) peak caller (13), customized by replacing the background control with the normal sample in each pair was also applied. SEACR reported a median of 4,483 peaks that were elevated in tumors, whereas when Tumor and Normal were exchanged only a median of 15 peaks elevated in normal tissue were identified, demonstrating thatAttorney Docket No.1426.48.WO RNAPII hypertranscription is more common than RNAPII hypotranscription in these cancer samples.

[0124] We next asked whether SEACR Tumor-versus-Normal peak calls corresponded to the 100 top cCREs ranked by T-N in the overall list representing all seven tumors. All 100 cCREs at least partially overlapped one or more SEACR Tumor / Normal peak calls, and in addition, the large majority of the 100 top-ranked cCREs intersected with overlapping SEACR peak calls from multiple Tumor / Normal pairs (Table 1). Each of the #1-ranked cCREs in the breast, colon, liver, lung and rectum tumor samples respectively intersected MSL1, RFFL, PABPC1, CLTC and SERINC5 genes and overlapped SEACR peak calls in 4-5 of the 7 tumors (FIG. 1N-1S, FIGS. 10A-10D). It was concluded that the most strongly RNAPII-hypertranscribed regulatory elements tend to be strongly hypertranscribed in multiple human cancers of different types, including liver cancers from different individuals (FIGS.11A-11F).

[0125] A much lower level of mitochondrial DNA (mtDNA) was observed in most tumor samples than in their matched normal samples for both mouse and human (FIG.12A-12B), suggesting that these tumors contain fewer mitochondria. To test this interpretation, publicly available ATAC-seq data from both the TCGA and ENCODE projects was mined, and similar reductions in cancer samples was observed (FIG.12C-12D). Such reductions in mtDNA have been reported based on whole-genome sequencing (14). HER2 amplifications with selective sweeps

[0126] All but one of the top 25 cCREs are located on Chromosome 17 (Table 1). Eight of these cCREs are within Chr17q12 and 13 are within Chr17q21, each spanning a few hundred kilobases in length in the breast and colon tumors not seen in the normal tissue (FIG.2A-2B, FIGS.13A- 13L). High RNAPII occupancy over the cCREs in Chr17q12-21 can account for most of the RNAPII hypertranscription signal in the breast and colon samples, centered over the ERBB2 gene (FIGS. 14A-14H). ERBB2 encodes human epidermal growth factor receptor 2 (HER2), and is commonly amplified in cancer and is a target of breast cancer therapy (15).

[0127] To confirm that the broad regions of RNAPII enrichment around the ERBB2 promoter correspond to HER2 amplifications in the breast and colon patient samples, we applied SEACR, which densely tiled a ~150-kb region centered over the ERBB2 promoter (FIG. 2A-2B). To ascertain whether dense tiling using SEACR can detect amplification events, SEACR broad peaks were called on published K562 RNAPII-Ser5p CUTAC datasets (16, 17). A single region heavilyAttorney Docket No.1426.48.WO tiled with broad SEACR peaks corresponding to an annotated amplification specific for K562 cells was observed. Zooming in revealed that the end of the densely tiled region corresponded precisely to the t(9;22)(q34;q11) translocation breakpoint of BCR-ABL (FIG. 15A-15B), which was confirmed by observing a broad SEACR peak on the ABL1 side of the translocation breakpoint (FIG. 15C). Thus, our approach using RNAPII-Ser5p CUTAC and SEACR can identify and precisely map regional amplifications such as are found in tumors with BCR-ABL and HER2 amplifications.

[0128] To delineate possible RNAPII hypertranscription features within Chr17q12-21, successive 1-kb tiles were binned over each 1-Mb region centered on the highest peak, corresponding to the ERBB2 promoter in Chr17q21 and the RFFL promoter in Chr17q12, and plotted count density within each bin with curve-fitting and smoothing. Multiple broad summits appeared in both breast and colon tumor-versus-normal tracks, and the six summits in the breast tumor sample accounted for the six highest ranked Chr17 promoter peaks (FIG.2C-2D). Count densities of the four highest ranking cCREs outside of Chr17q12-21 (Table 1), but tumor peaks in these regions were at least an order-of-magnitude lower than the ERBB2 peaks in the breast and colon tumor samples (FIG. 2E-2H). Of the six summits in the breast tumor sample, ERBB2 and MSL1 also appeared in the colon tumor sample, whereas no other samples showed prominent summits above normal in Chr17q12-21 (FIG.2C-2D). MSL1 encodes a subunit of a histone H4-lysine-16 acetyltransferase complex required for upregulation of the mammalian X chromosome (18).

[0129] Each of the six summits in the Chr17q12-21 region in the breast tumor sample were superimposed over the genomic tracks on expanded scales for clarity, centered over the highest promoter peak in the region (FIG.2I). For ERBB2, the ~100-kb broad summit is almost precisely centered over the ~1-kb wide ERBB2 promoter peak. Although the other summits are less broad, each is similarly centered over a promoter peak. Insofar as there are multiple summits much broader than the promoter peaks that they are centered over, the results are inconsistent with independent upregulation of promoters over the HER2-amplified regions. Rather, it appears that a HER2 amplification event was followed by clonal selection for broad regions around ERBB2 and other loci within each amplicon, consistent with the observation of clonally heterogeneous HER2 amplifications in primary breast tumors by whole-genome sequencing (19). Clonal selection may be driven by selective sweeps (20) following amplification events that generate extrachromosomal DNA in double-minute acentric chromosomes, which partition unequally during each cell divisionAttorney Docket No.1426.48.WO (21-23). Such copy number gains within a tumor can result in intra-tumor heterogeneity (23, 24) and are potential factors for resistance to therapy (25). FFPE-CUTAC thus potentially provides a general diagnostic strategy for detection and analysis of amplifications and clonal selection during cancer progression and therapeutic treatment.

[0130] One of the summits in the breast tumor sample absent from the colon tumor sample corresponds to the bidirectional promoters of MED1 and CDK12, both of which have been shown to functionally cooperate with co-amplified ERBB2 in aggressive breast cancer (26, 27). CDK12 is the catalytic subunit of the CDK12 / Cyclin K kinase heterodimer complex, which phosphorylates RNAPII for productive transcriptional elongation (28, 29). As Cyclin K is the regulatory subunit of the CDK12 kinase, we would expect that the CCNK gene that encodes Cyclin K would be strongly upregulated in the breast tumor but not necessarily in the colon tumor. Indeed, a 5.4-fold increase in RNAPII-S5p was seen over the CCNK promoter in the breast tumor relative to adjacent normal tissue, whereas in the colon tumor there is a 2.1-fold increase (FIG.2J), and a 0.9-2.5-fold increase for the other five tumors, consistent with RNAPII hypertranscription driven in part by CDK12 amplification in this tumor. RNAPII over histone genes predicts aggressiveness in meningiomas and breast tumors

[0131] To evaluate how effectively FFPE-CUTAC can resolve differences between the seven tumor samples, a cCRE-based UMAP was constructed including 114 individual human datasets. Whereas normal samples produced mixed clusters, tumor samples formed tight homogeneous clusters separated by tissue type (FIG. 3A). This implies that paused RNAPII at regulatory elements is more discriminating between tumors than between the tissues that the tumors emerge from. Relatively few samples and shallow sequencing depths were needed for tight clustering (FIG.3B).

[0132] As the exceptionally S-phase-dependent histone loci are expressed in proportion to the amount of replicated DNA, we wondered whether cancer cell hypertranscription functions to increase engaged RNAPII at these loci to load up on histones at S-phase for more rapid cell proliferation. For both the mouse and human clustered histone genes differences between tumor samples were observed consistent with RNAPII hypertranscription at cCREs differing between samples (FIGS.16A-16B). If histone production at S-phase is rate-limiting for proliferation, then a correlation between cancer aggressiveness and RNAPII, specifically over histone genes would be observed. As a test of this hypothesis, FFPE-CUTAC was applied to 30 meningioma patientAttorney Docket No.1426.48.WO samples and a UMAP constricted that also included the Tumor-Normal pairs described above. Using RNAPII abundance within cCREs, it was found that the meningiomas clustered separately from other tumors and from normal (FIG. 3C-3D). When the same UMAP construction was performed using only RNAPII abundance over the 64 S-phase-dependent histone genes, it was found that all tumors regardless of type clustered together in a cline that overlapped normal at one end (FIG.3E). This suggested that RNAPII at histone genes can distinguish cancer from normal but is insensitive to cancer type differences. In a double-blind test of whether the cline overlapping normal in multi-dimensional space is an indication of tumor aggressiveness, the 15 samples were rank-ordered with WHO grades based on distance from normal samples for RNAPII occupancy in 500-bp bins, cCREs, histone genes or ribosomal protein genes (FIG.3F). Best performance was for the histone genes (r=0.57, p<0.05), consistent with the hypothesis that high RNAPII levels at histone genes drive proliferation in cancer.

[0133] To determine whether S-phase-dependent histone genes can predict cancer aggressiveness in an invasive tumor type, we performed FFPE-CUTAC on a set of 10-μm breast tumor FFPEs from 13 patients representing three major subtypes. When we included these samples with the seven diverse tumor and normal samples (FIG.1F-1L) and used cCREs for UMAP construction, tight clustering was observed according to tumor type (FIG.3G). However, when histone genes were used for UMAP construction, a single large cluster was observed at one end of a cline that overlapped normal at the other end (FIGS. 3H-3I). We conclude that histone genes alone can predict cancer aggressiveness in both non-invasive meningiomas and multiple invasive breast cancer subtypes. RNAPII over histone genes accurately predicts rapid recurrence in meningiomas

[0134] WHO grade is a coarse predictor of recurrence (30), so to predict recurrence for each patient sample, FFPE-CUTAC data was integrated with RNA-seq data using canonical correlation analysis in multi-dimensional space, based on normalized counts over each RefSeq-annotated human gene (FIG.17). A gene was defined as spanning from the 3’-most transcript end through the 5’-most end, stopping when either end of the next gene or LINE element is reached. Near- coincidence for 17 of 19 matched RNA-seq and FFPE-CUTAC samples as observed.

[0135] To predict meningioma patient recurrence, FFPE-CUTAC samples were classified by the histone genes signal and used the top 20 RNA-seq nearest neighbors to compare the patient recurrences (FIG. 4A, FIG. 18A). Indeed, a highly significant association between high histoneAttorney Docket No.1426.48.WO signals in FFPE-CUTAC samples and rapid patient recurrence was found (p<10-8, FIG. 4B left, FIG.18B). Using only the normalized FFPE-CUTAC counts at the 64 histone the five most rapidly recurring were distinctly separated from the 25 otheraligns with the generally low recurrence rate of meningiomas, and are predominantly benign (31). In contrast, levels of RNAPII over ribosomal protein genes or mtDNA failed to significantly separate rapidly recurring from benign, regardless of the thresholds applied (FIG.4B, FIGS.18C-18D). Although significant separation of rapidly recurring from benign were observed using Chr22q, the most frequently lost whole-arm (30) (FIG. 4B right), and significant separation for Chr1q gains and Chr6p losses (FIGS. 18E-18F), the levels of significance were much lower than for separation using RNAPII at the histone genes. This finding that high levels of RNAPII over RC histone genes accurately predicts poor outcomes in meningioma could imply a causal basis. RNAPII over histone genes predicts whole-arm chromosome losses

[0136] As a positive control for separation of malignant from benign, total aneuploidies were counted from RNA-seq data and observed best separation for five malignant and 25 benign tumors, closely matching our prediction based on RNAPII at histone genes over the range of 3-7 malignant (FIG.5A). This led us to ask whether there is a relationship between overproduction of histones and total aneuploidy by plotting RNAPII levels over the histone genes for each patient as a function of the total number of whole-arm chromosome gains or losses. A weak non-significant positive Spearman correlation with gains was observed (p<0.2), but a highly significant correlation with losses (p<0.006) (FIG. 5B). To test whether this excess of losses over gains applied to all chromosome arms, we asked whether for each patient, there was a net increase or decrease in the level of RNAPII over histone genes. Indeed, 38 / 39 autosomal arms showed a net positive correlation for the meningioma patient population (FIG.5C, FIG.19A), suggesting that RNAPII at histone genes predicts whole-arm losses in meningioma. This excess of whole-arm losses was observed for the breast cancer samples, where we also observed excesses of losses over gains for 38 / 39 autosomal whole-arm aneuploids (FIG. 5C, FIG. 19B), as expected if overexpression of histones drives both over-proliferation and whole-arm chromosome losses in cancer. Discussion

[0137] Elevated RNAPII over genes and regulatory elements was demonstrated as a direct measure of hypertranscription in diverse human cancers and identifies and precisely maps amplifications and selective sweeps in small clinical samples. Likewise, a close correspondenceAttorney Docket No.1426.48.WO between high RNAPII at the 64 S-phase-dependent histone genes was observed consistent with cytological evidence of exceptionally high levels of RNAPII at mouse histone genes (4) and both RNAPII (2, 3) and Myc (32) at Drosophila histone locus bodies exclusively during S-phase. This led to the hypothesis that the single functional role of hypertranscription in cancer is to produce enough histones to keep up with the requirement for packaging new DNA in cancer cells to proliferate faster than normal cells. This prediction was confirmed by performing FFPE-CUTAC on a set of 30 human meningiomas and showing that RNAPII at the 64 S-phase-dependent histone genes, which comprise only 1 / 100,000thof the human genome, successfully estimated WHO grade and accurately predicted rapid recurrence, which corresponds to elevated expression of proliferation genes (30). Although meningiomas are not invasive, elevated RNAPII at histone genes was also observed in invasive breast tumors. The ability of RNAPII FFPE-CUTAC at only the 64 human genes to predict aggressiveness in a common intracranial tumor and in multiple breast cancer subtypes implies that a rapid PCR assay for histone gene RNAPII or transcription (33) may become an inexpensive general cancer diagnostic tool, revolutionizing precision oncology.

[0138] The levels of RNAPII at the S-phase-dependent histone genes correlated with total aneuploidies was also found, which are present in nearly all meningioma patient samples (30), consistent with the occurrence of ~90% whole-arm imbalances in pan-cancer TCGA data (34). Whole-arm losses were observed to be in excess of gains for 38 of the 39 autosomal arms for both meningiomas and breast tumors representing multiple subtypes. To explain how overproduction of histones might account for this striking whole-arm loss bias in patient tumors, we propose that excess H3 histones compete with CENP-A histones at S-phase for nucleosome assembly at centromeres (35, 36) (FIG. 5D). Production of S-phase histones is tightly regulated (37) and overproduction in cancer and displacement of CENP-A nucleosomes are known to result in the generation of DNA–RNA hybrids, likely due to transcription–replication conflicts causing delayed DNA replication, centromere breakage and loss of whole chromosome arms (38, 39). Thus, RNAPII excess over histone genes at S-phase provides a mechanistic basis for understanding not only how cancer cells can proliferate faster than their neighbors but also how this process might generate centromeric breaks and whole-arm imbalances that drive most cancers. Table 1. Ranked cCREsAttorney Docket No.1426.48.WO # Multi # w / Cancer Tumor Gene_ ple SEACR SEACR public Encoded Rank CCRE T e Chr Start End name hits eaks eaks ations Protein ger E g ipi esi or se 1 nt ns ip x y D 1 as ry B - nt 2 nsAttorney Docket No.1426.48.WO Fibronectin type-III Domain- EH38E185 FNDC Containing e ke mb n 1 de ul 1 tin er 2, er r 2 ic onAttorney Docket No.1426.48.WO EH38E208 23257284 23257319 Prothymosi 36 3049-prom Li chr2 76 PTMA St 1 64ne Alpha Homocystei n in nt as ry B er30e ag iat d er pe ne ear eo f 2 ha ns yl 1 re 06 nd 2Attorney Docket No.1426.48.WO Zinc Finger And BTB Br, Co, 24 Domain EH38E143 24421152 24421187 ZBTB Li Lu Containing 3- ge pto ge n ck 1 on d me t B2 g nt nd 3Attorney Docket No.1426.48.WO Receptor Alpha Br, Co, M-Phase EH38E165 12371738 12371753 MPHO Li L 5 Ph h pro al 3a ic on 1 r er - se nd E3 ne nd AS g 2 g ti 4 iat veAttorney Docket No.1426.48.WO DnaJ Heat Shock Protein Br Co 28 Famil C3 - e, ti 1 C d e 5 d 2Table 2. Replication-coupled Histones ChromoStart End GeneStr somename LengthandHistone name Statusd d d d al d dAttorney Docket No.1426.48.WO chr1 149832329 149832725 NM_001034077_chr1_149832329395 - HIST2H4B Reviewedd d d d al d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d dAttorney Docket No.1426.48.WO chr6 27798951 27799305 NM_003541 353 - HIST1H4K Reviewed chr6 27805657 27806117 NM_003510 459 - HIST1H2AK Reviewed hr6 27806439 27806888 NM 003520 448 + HIST1H2BN R vi w d d d d d d d d1. M. Zatzman et al., Widespread hypertranscription in aggressive human cancers. Sci Adv 8, eabn0238 (2022). 2. F. Lu et al., Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription. bioRxiv doi: 10.1101 / 2023.10.07.561364, (2024). 3. S. K. Huang, P. H. Whitney, S. Dutta, S. Y. Shvartsman, C. A. Rushlow, Spatial organization of transcribing loci during early genome activation in Drosophila. Curr. Biol.31, 5102 (2021). 4. D. B. Mahat et al., Single-cell nascent RNA sequencing unveils coordinated global transcription. Nature 631, 216 (2024). 5. R. Milo, R. Phillips, Cell Biology by the numbers. (Garland, 2016). 6. S. Henikoff et al., Epigenomic analysis of Formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun 14, 5930 (2023). 7. J. E. Moore et al., Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583, 699 (2020). 8. T. Ozawa et al., A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-kappaB. Cell Rep 23, 3787 (2018). 9. F. Szulzewsky et al., Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis. Genes Dev.34, 1051 (2020). 10. C. Dai et al., PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev.15, 1913 (2001). 11. D. G. Altman, J. M. Bland, Measurement in medicine: the analysis of method comparison studies. The Statistician 32, 307 (1983). 12. S. Patange et al., MYC amplifies gene expression through global changes in transcription factor dynamics. Cell Rep 38, 110292 (2022). 13. M. P. Meers, D. Tenenbaum, S. Henikoff, Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics Chromatin 12, 42 (2019). 14. E. Reznik et al., Mitochondrial DNA copy number variation across human cancers. eLife 5, (2016). 15. H. Zhang et al., HER2 evaluation for clinical decision making in human solid tumours: pearls and pitfalls. Histopathology doi:10.1111 / his.15170, (2024). 16. S. Henikoff, J. G. Henikoff, K. Ahmad, Simplified Epigenome Profiling Using Antibody- tethered Tagmentation. bio-protocol 11, e4043 (2021).Attorney Docket No.1426.48.WO 17. D. H. Janssens et al., CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells. Genome Biol 23, 81 (2022). 18. X. Deng et al., Mammalian X upregulation is associated with enhanced transcription initiation, RNA half-life, and MOF-mediated H4K16 acetylation. Dev Cell 25, 55 (2013). 19. Y. Fan et al., Characteristics of DNA macro-alterations in breast cancer with liver metastasis before treatment. BMC Genomics 24, 391 (2023). 20. D. Yang et al., Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution. Cell 185, 1905 (2022). 21. X. Yan, P. Mischel, H. Chang, Extrachromosomal DNA in cancer. Nat Rev Cancer 24, 261 (2024). 22. R. J. Kaufman, P. C. Brown, R. T. Schimke, Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc. Natl. Acad. Sci. U. S. A.76, 5669 (1979). 23. P. C. Nowell, The clonal evolution of tumor cell populations. Science 194, 23 (1976). 24. J. R. M. Black, N. McGranahan, Genetic and non-genetic clonal diversity in cancer evolution. Nat Rev Cancer 21, 379 (2021). 25. D. L. Schaff, A. J. Fasse, P. E. White, R. J. Vander Velde, S. M. Shaffer, Clonal differences underlie variable responses to sequential and prolonged treatment. Cell systems 15, 213 (2024). 26. M. Forster-Sack et al., ERBB2-amplified lobular breast carcinoma exhibits concomitant CDK12 co-amplification associated with poor prognostic features. The journal of pathology. Clinical research 10, e12362 (2024). 27. M. Marotta et al., Palindromic amplification of the ERBB2 oncogene in primary HER2- positive breast tumors. Sci Rep 7, 41921 (2017). 28. B. Bartkowiak et al., CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev.24, 2303 (2010). 29. M. Tellier et al., CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation. Nucleic Acids Res 48, 7712 (2020). 30. H. N. Thirimanne et al., Meningioma transcriptomic landscape demonstrates novel subtypes with regional associated biology and patient outcome. Cell genomics 4, 100566 (2024). 31. S. Goebel, H. M. Mehdorn, Development of anxiety and depression in patients with benign intracranial meningiomas: a prospective long-term study. Support. Care Cancer 21, 1365 (2013). 32. K. Daneshvar, A. Khan, J. M. Goodliffe, Myc localizes to histone locus bodies during replication in Drosophila. PLoS One 6, e23928 (2011). 33. R. Sun, H. Qi, Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis. Gene expression patterns : GEP 14, 30 (2014). 34. J. Shih et al., Cancer aneuploidies are shaped primarily by effects on tumour fitness. Nature 619, 793 (2023). 35. C. C. Chen et al., Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription. Dev Cell 34, 73 (2015). 36. M. D. Blower, B. A. Sullivan, G. H. Karpen, Conserved organization of centromeric chromatin in flies and humans. Developmental Cell 2, 319 (2002).Attorney Docket No.1426.48.WO 37. W. F. Marzluff, R. J. Duronio, Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences. Curr. Opin. Cell Biol.14, 692 (2002). 38. S. Giunta et al., CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy. Proc. Natl. Acad. Sci. U. S. A.118, (2021). 39. A. Scelfo et al., Specialized replication mechanisms maintain genome stability at human centromeres. Mol. Cell 84, 1003 (2024). 40. S. Henikoff, Aligned files for Henikoff et al.2025 RNA Polymerase II at histone genes predicts outcome in human cancer. Zenodo doi: 10.5281 / zenodo.13138686. (2025). 41. S. Henikoff, Custom code for Henikoff et al.2025 RNA Polymerase II at histone genes predicts outcome in human cancer. Zenodo doi:10.5281 / zenodo.14649635, (2025). 42. D. Hambardzumyan, N. M. Amankulor, K. Y. Helmy, O. J. Becher, E. C. Holland, Modeling Adult Gliomas Using RCAS / t-va Technology. Translational oncology 2, 89 (2009). 43. A. S. Hinrichs et al., The UCSC Genome Browser Database: update 2006. Nucleic Acids Res 34, D590 (2006). 44. A. R. Quinlan, BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Current protocols in bioinformatics 47, 11121 (2014). 45. M. Martin, Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads. EMBnet.journal 17 doi:10.14806 / ej.17.1.200, (2010). 46. B. Langmead, S. L. Salzberg, Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357 (2012). 47. P. Danecek et al., Twelve years of SAMtools and BCFtools. Gigascience 10, (2021). 48. T. Stuart et al., Comprehensive Integration of Single-Cell Data. Cell 177, 1888 (2019). 49. A. Serin Harmanci, A. O. Harmanci, X. Zhou, CaSpER identifies and visualizes CNV events by integrative analysis of single-cell or bulk RNA-sequencing data. Nat Commun 11, 89 (2020). 50. V. E. Clark et al., Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas. Nat. Genet.48, 1253 (2016). Example 2. PCR Evaluation for Cancer Aggressiveness

[0140] An overview of an exemplary general PCR test for cancer aggressiveness is shown in FIG.20. A strategy for assaying histone hypertranscription by PCR is described by the following example steps: Total nucleic acids from a biological specimen are solubilized using a Chaotropic buffer such as in the Thermo-Fisher NucleoSpin kit and bound to NucleoSpin column. DNA is first eluted using the NucleoSpin RNA / DNA Buffer set designed for the NucleoSpin kit. A 50 nt spike-in oligonucleotide with primer-complementary sequences at either end is added and both the cDNA and spike-in are amplified by PCR or qPCR using histone gene-specific primers. Meanwhile, the filter is then treated with DNAase, and the RNA is eluted and reverse-transcribed using the histone-specific anti-sense primer. After (optional) RNaseH treatment, the spike-in oligonucleotide is added followed by PCR or qPCR amplification using histone gene-specific primer. Histone gene hypertranscription isAttorney Docket No.1426.48.WO calculated as the ratio of cDNA to genomic DNA using the cDNA:gDNA ratio of the spike-in oligo as an internal calibration standard.

[0141] Nested PCR primers for histone gene RNA:DNA PCRs. A) To design PCR primers that amplify all histone mRNAs, the most conserved regions of a Clustal-W multiple alignment are identified within coding sequence and primers with similar melting temperatures are chosen. An example of nested primers is shown with two forward and one reverse primer, providing increased specificity (FIG. 21A). Invariant (*) and Conserved (.) residues are indicated below the alignment. Primers and spike-in oligonucleotide sequences are shown in FIG. 21B with predicted melting temperatures for each of the five RC histones. Consensus and degeneracy rules follow CODEHOP recommendations (Rose et al. Nucl. Acids Res.26:1628-1635, 1998). As hypertranscription is a global phenomenon, using an endogenous gene as a standard would not serve as a useful control, as it would be expected to show changes in RNA levels similar to those for histone genes. Therefore, an amplifiable spike-in 50 nt oligonucleotide resolvable by Tapestation analysis from the histone cDNA products can be utilized. This avoids mis-interpreting assay failure from hypotranscription and provides a standard that can be matched to the high level of expected histone product. H1 83 bp H1-5u 5'-GGCTAYGAYGTRGAGAARAA-3' (SEQ ID NO:1) 56-67°C H1-5p 5'-AACAGCCGCATCAAGCT-3'(SEQ ID NO:2) 65°C H1-3p 5'-CCTTCTTGTTGAGYTTRAAGGA-3'(SEQ ID NO:3) 60-65°C H1si50 5’-AACAGCCGCATCAAGCTGTTTAAACGCGTCCTTYAARCTCAACAAGAAGG- 3'(SEQ ID NO:4) Forward primer PmeI Reverse primer H2A 137 bp H2A-5p 5'-GAGCTGGCNGGCAA-3' (SEQ ID NO:5) 62-66°C H2A-3p 5'-GGCARRACRCCRCCCT-3' (SEQ ID NO:6)64-72°C H2A-3d 5'-ACGGCCTGGATGTT-3' (SEQ ID NO:7) 59°C H2Asi50 5'-GAGCTGGCNGGCAATAGGGATAACAGGGTAATTCAGGGYGGYGTYYTGCC- 3'(SEQ ID NO:8) Forward primer I-SceI Reverse primer H2B 93 bp H2B-5p 5'-AACAAGCGCTCGACCAT-3'(SEQ ID NO:9)65°C H2B-5u 5'-CTGCCCGCCTGGC-3' (SEQ ID NO:10) 68°C H2B-3p 5'-TACTTGGTGACGGCCTT-3'(SEQ ID NO:11) 64°C H2Bsi50 5'-CTGCCCGCCTGGCATTAGGGATAACAGGGTAATAAGGCCGTCACCAAGTA- 3'(SEQ ID NO:12) Forward primer I-SceI Reverse primerAttorney Docket No.1426.48.WO H3 116 bp H3-5p 5'-GTGATGGCGCTGCA-3' (SEQ ID NO:13)64°C H3-5u 5'-GAGGCYWGCGAGGC-3' (SEQ ID NO:14)62-67°C H3-3p 5'-CCACGGATCGCCGC-3' (SEQ ID NO:15)63°C H3si50 5'-GTGATGGCGCTGCATAGGGGATCATAACAGGGTAATGCGGCGATCCGTGG- 3'(SEQ ID NO:16) Forward primer I-SceI Reverse primer H4 89 bp H4-5p 5'-TCATCTACGAGGAGACYCG-3' (SEQ ID NO:17)62-65°C H4-3p 5'-GCGYTTGGCGTGCTC-3' (SEQ ID NO:18) 65-68°C H4-3d 5'-CGTANACCACATCCATGGC-3' (SEQ ID NO:19) 62-66°C H4si50 5'-TCATCTACGAGGAGACYCGGAATTCGTTTAAACTCGAGCACGCCAARCGC- 3'(SEQ ID NO:20) Forward primer EcoRI PmeI XhoI Reverse primer Example 3. Total whole-arm chromosome losses predict malignancy in human cancer

[0142] As shown herein, whole-arm losses predict recurrence in RNA-seq, DNA-seq and pan-cancer data, supporting and extending the model that centromere breaks caused by histone overexpression initiate aneuploidy, and showing that centromere breaks suffice without mitotic segregation errors to explain most aneuploidy in cancer.

[0143] Gain or loss of whole chromosome arms following centromere breaks is frequent in cancer, but whether or not there is a common initiating event is unknown. The following working example shows that the total number of whole-arm losses predicts patient outcomes across cancer types, suggesting a causal relationship. This general excess of losses over gains is not predicted by mitotic error models of aneuploidy but rather suggests that centromere breaks themselves initiate whole-arm aneuploidies. Insofar as aneuploidy reshapes the selective landscapes that drive most cancers, these results have clinical implications.

[0144] Aneuploidy is a familiar hallmark of cancer that was first described well over a century ago (1). In 1890, David Hansemann observed asymmetric mitoses in a variety of epithelial cancers but not in normal tissues. Among the forms that Hansemann illustrated were examples of chromatids away from the metaphase plate that were either attached or unattached to the mitotic spindle, in addition to many examples of multipolar spindles. More than 130 years after Hansemann’s observations, the causes of aneuploidy have been understood as belonging to any of four classes of mitotic errors (2): merotelic attachments, where a single kinetochore connects to opposite spindles, resulting in a chromosome that remains at the metaphase plate; extra centrosomes, where chromosomes segregateAttorney Docket No.1426.48.WO to three or more poles; unattached kinetochores, where only one sister chromatid is attached and both are pulled to the same pole; or cohesion defects where sister chromatids either release from one another prior to anaphase or fail to release at anaphase. Notably, each of these chromosome instability mechanisms can result in the gain or loss of chromosomes or chromosome fragments based on cytological preparations of tumors. This diversity of mechanisms that are thought to drive aneuploidy in cancer severely complicates therapeutic strategies.

[0145] In recent years, whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) have provided efficient alternatives to karyotype analysis for scoring aneuploidies in cancer patient samples. Whole-chromosome, whole-arm and partial gains or losses can be accurately scored by measuring differences in DNA or RNA abundances across the genome. These genomic and transcriptomic studies have led to the realization that aneuploidy encompasses multiple varieties of somatic copy number alterations generated by different molecular mechanisms (3). Whole-arm aneuploidy is especially common in cancer and certain losses or gains are important prognostic indicators. For example, in myelodysplastic syndrome, which can lead to acute myeloid leukemia, loss of chromosome arm 5q predicts a more positive outcome, while loss of chromosome 7 or arm 7q predicts a more negative outcome after bone marrow transplantation, and these indicators have long been used to determine the course of treatment (4).

[0146] WGS or RNA-seq of routine patient samples is often challenging because clinical samples are typically banked as formalin-fixed paraffin-embedded sections (FFPEs), but RNA Polymerase II (RNAPII) profiling in FFPEs as an efficient method for assessing chromatin profiling in clinical samples was recently implemented (5). A variety of patient tumor and normal samples were also surveyed, including 30 meningiomas and 15 breast tumors. Using the RNAPII signal at candidate cis- regulatory elements (cCREs) from the Encyclopedia of DNA Elements (ENCODE) to identify whole- arm aneuploids, it was found that the total number of whole-arm aneuploids accurately predicted rapid recurrence in meningiomas (6). RNAPII at S-phase-dependent histone genes strongly correlated with whole-arm losses relative to gains in both meningiomas and breast tumors, suggesting a causal relationship.

[0147] Here, available public RNA-seq and WGS datasets were used to test the generality of predictions based on total arm aneuploid counts using RNAPII data. These analyses confirmed that the total number of whole-arm losses predicts outcome better than gains. It is proposed that whole- arm losses are immediate consequences of most centromere breaks, and others with partially intactAttorney Docket No.1426.48.WO centromeres form micronuclei, where they undergo S-phase replication and reattachment at a subsequent anaphase. Whole-arm losses correlate with recurrence in meningioma RNA-seq and pan-cancer WGS data.

[0148] Whole-arm losses and gains of metacentric chromosomes are generated by breaks in centromeres or pericentromeric regions, although the underlying mechanisms have been speculative (7). We previously showed that RNAPII at histone genes in 30 patient samples predicted rapid recurrence when integrated with RNA-seq data from 1298 meningiomas. We also showed that RNAPII at histone genes correlated with whole-arm losses relative to whole-arm gains, and we wondered whether the meningioma RNA-seq data shows the same bias of losses over gains. S-phase- dependent histone mRNAs are the only RNAPII-transcribed protein-coding genes that are not 3’- polyadenylated, and so they are grossly under-represented in RNA sequencing datasets (FIG.26).

[0149] To determine whole-arm gains and losses in RNA-sequencing (RNA-seq) data, CaSpER was leveraged to identify the copy number variants (CNVs) and, hence, determine the whole-arm gains or losses according to the consistency-based large-scale CNV calls (8, 9). We observed very low levels of recurrence with <3 whole-arm losses, intermediate levels of recurrence with 3-6 losses and highest levels of rapid recurrence with >6 losses (FIG.22A, left panel). Low levels of recurrence were also observed for <3 gains, however, >6 gains were not associated with rapid recurrence (FIG.22A, middle panel). Total whole-arm aneuploidy also predicted recurrence, although not as well as whole-arm losses (FIG.22A, right panel).

[0150] The highly significant loss over gain bias in meningiomas that we observed in RNA-seq data encouraged us to ask whether the same bias can be detected in The Cancer Genome Atlas Project (TCGA) data. Examination of pan-cancer whole-genome sequencing (WGS) data from TCGA for 10,522 patients (10) revealed an overall average excess of whole-arm losses per patient (5.8) over gains (4.2) (Table 3). Table 3. Overall across 10,522 patient samples ofAttorney Docket No.1426.48.WO Per patientLoss 5.78 0.148 0.162n8 2 9 2 n 3 2 3 8 n 9 6 1 1 n 5 6 9Attorney Docket No.1426.48.WO Gain 2.05 0.12 0.07 Gain 1.26 0.07 0.05 n 6 5 7 6 n 4 5 5 6 n 4 0 7 7 n 2 3 8 6 n 8 3Attorney Docket No.1426.48.WO Loss 6.36 0.16 0.11 Loss 5.78 0.15 0.11 Gain 4.06 0.1 0.08 Gain 3.54 0.09 0.08 n 1 0 7 6 n 8 6 5 4 n 3 5 2 3 n 9 7 2 1 n 4 4Attorney Docket No.1426.48.WO Loss 0.49 0.03 0.04 Loss 0.25 0.01 0.01 Gain 1.66 0.1 0.09 Gain 0.27 0.02 0.01 n 4 5 5 .1 n 5 1 .1 7 n 4 8 4 1 n 4 8 .1 7 n 0 9Attorney Docket No.1426.48.WO Per patient Per patientLoss 6.48 0.17 0.13 Loss 7.11 0.18 0.13 11 n 8 5 .1 8 n 1 4 4 8 n 5 4 8 6 n 2 0 1 6 n 5 5Attorney Docket No.1426.48.WO Per patient Per patientLoss 1.14 0.07 0.05 Loss 4.63 0.27 0.16 03 n 3 3 1 1 n 1 2 0 0 n 4 4 4 4 n 3 3 3 3 n 6Attorney Docket No.1426.48.WO Gain 751 19.26 11 Gain 1462 37.49 28 Per patient Per patient07 5 n 7 2 3 4 n 4 9 5 6 n 5 7 9 2 n1415n0Attorney Docket No.1426.48.WO Gain 71 4.18 3Per patient04

[0151] To predict outcome based on wholewe used recurrence (disease-free interval) data from a previous study (11), grouping by the same whole-arm aneuploid frequency intervals as for meningiomas. Strikingly, we observed qualitatively similar predictions for losses, gains and total aneuploids for pan-cancer data as for meningiomas, with the most rapid recurrence for >6 losses but not for >6 gains (FIG. 22B). This qualitative concordance between pan-cancer WGS data and meningioma RNA-seq data predictions of clinical outcome is especially notable considering the large variations in the average number of losses and gains between individual cancer types (FIG.22C). Whole-arm are more frequent than whole-chromosome aneuploids across cancer types.

[0152] Based on breakpoint analysis, whole-arm aneuploids account for 23 of the 25 most frequent breakage events and span on average 22.5% of the human genome in TCGA pan-cancer data (12). When whole-arm gains and losses are plotted as a fraction of the total for each cancer type, the overall frequencies are seen to vary over a wide range (FIG. 23, FIG. 27). For example, adrenocortical carcinoma (ACC) averages 9.3 gains and 9.0 losses per patient, whereas acute myeloid leukemia (LAML) shows only 0.85 gains and 0.72 losses per patient. Arm-to-arm differences are also cancer type-specific, with very similar frequencies for all 39 autosomal arms for ACC but conspicuous arm- to-arm variations for glioblastoma (GBM). It is generally assumed that concordant p and q arm loss numbers in genomic data represent whole-chromosome losses. Indeed, a molecular criterion for glioblastoma is gain of Chromosome 7 and loss of Chromosome 10 (13) with 7p and 7q gains and 10p and 10q losses in ~80% of tumors compared with ~10% total gains and losses for most other arms (FIG.23). Based on concordance between p and q arms within a tumor, TCGA patient data revealed that overall 21.6% of the total are whole-chromosome aneuploids, and these show a small overall excess whole-chromosome losses over gains (1.4 losses versus 1.2 gains, (Table 3). Therefore, despite examples such as GBM, which is driven primarily by Chr7 gain and Chr10 loss (14), whole-arm gains and losses are by far more conspicuous across cancer types in TCGA. Metacentric and acrocentric aneuploidies occur at similar frequencies across cancer types.Attorney Docket No.1426.48.WO

[0153] In cancer, breakpoints in centromeric and pericentric regions are on average 4.4 times more frequent than breakpoints in euchromatic arms based on breakpoint density along the chromosome (12). Because these regions consist of tandemly repetitive a-satellite DNA and the functional centromere accounts for only ~5% of the total (15), the 4.4-fold excess of centromere-specific breaks is likely a gross under-estimate of the likelihood that a break in a functional centromere will result in a whole-arm aneuploid. As the vast majority of aneuploid chromosomes in cancer must have undergone centromere breakage, and this alone may result in failure to attach to the mitotic spindle, it is possible that aneuploidy occurs without any other mitotic error.

[0154] To determine whether mitotic errors, in addition to centromere breaks, are responsible for whole-arm gains and losses in cancer, we took advantage of the two classes of human chromosomes based on the position of the centromere. Metacentric chromosomes have two euchromatic arms (FIG. 24A) and so require a centromere break to generate a whole-arm aneuploidy (FIG.24C). There are five human acrocentric chromosomes (13, 14, 15, 21 and 22), which have similar kinetochore conformations as metacentrics (16), but only a single euchromatic arm (FIG.24B). Acrocentrics that are gained or lost by mitotic error will be effectively indistinguishable from those that have undergone a centromere break event (FIG. 24D). Acrocentric short arms comprise only redundant ribosomal DNA genes and other tandem repeats, and single short-arm gains or losses are not detected in genomic studies. Whole-chromosome meiotic or mitotic segregation errors occur, but centromere position does not predict frequency, based on pre-meiotic mitoses during human oocyte maturation (78% metacentrics expected, 83% observed, n = 52) (17). Thus, if mitotic errors are essential for the generation or perpetuation of a significant number of whole-arm aneuploids, then there should be an excess of acrocentrics (both those with and those without centromere breakpoints) relative to metacentrics (all of which must have centromere breakpoints).

[0155] To determine whether acrocentric are in excess over metacentric aneuploid chromosomes as expected for mitotic error in addition to centromere breaks, we have analyzed WGS data from TCGA for acrocentrics and metacentrics from 10,674 patients based on allele-specific copy number segmentation analysis across 33 cancer types. However, we observed no significant differences between acrocentrics and metacentrics in the frequency of whole-arm gains or losses or both (FIG. 24E, FIGS.28A-28C). We performed a similar analysis of long-read RNA-seq data from B-ALL and AML leukemias and again observed no significant differences (FIG. 29) (18). Considering that weAttorney Docket No.1426.48.WO are comparing all autosomal chromosome arms from >10,000 patient tumors, our inability to detect whole-arm events attributable to mitotic error is highly robust. Discussion Total whole-arm losses predict outcome across cancer types

[0156] In the previous study, it was had found that RNAPII at the S-phase-dependent histone genes is increased in most human cancers, reminiscent of the observation that elevated histone expression promotes life span extension in yeast (21). It was also shown that RNAPII at histone genes correlated strongly with total arm losses and speculated that histone overexpression results in centromere breaks that lead to aneuploidy (6). Here these findings were extended by showing that simply counting the number of whole-arm losses in public RNA-seq and WGS data predicts clinical outcome in diverse human cancers better than whole-arm gains. This is counter-intuitive given that trisomies (3 / 2 gain) occur in ~0.3% of newborns (22), but no autosomal monosomy (1 / 2 loss) is known to have ever come to term, and yet whole-arm losses are evidently more fit than gains in cancer (23, 24). As whole-arm aneuploids from metacentric chromosomes must be generated by centromere breaks, our finding that the degree of aneuploidy is patient-specific in general for all chromosome arms implies that whatever is causing centromere breaks is not chromosome-specific but rather is a general cellular event.

[0157] The relationship between aneuploidy and cancer has been vigorously debated ever since Boveri built on Hansemann’s careful observations with insights from genetics to argue that aneuploidy underlies the cancer phenotype (26). This debate has continued for over a century (27, 28), and recent evidence from TCGA data that whole-arm aneuploids result in either net gains of tumor drivers or losses of tumor suppressors (12) supports the Hansemann-Boveri hypothesis. In contrast, the question of whether mitotic errors cause aneuploidy has not been seriously challenged. Merotelic attachments of mouse chromosomes in cells with a Dido mutation were reported to break at anaphase (29), however, direct evidence for centromere breaks at anaphase is extremely limited. Furthermore, effective tension-dependent mechanisms have evolved to release attachments, sometimes resulting in lagging chromosomes (30). Therefore, in the absence of conclusive evidence that anaphase tension can break centromeric DNA of human chromosomes, we consider the generation of whole-arm aneuploids during mitosis to be unlikely to account for their high abundance in cancer. A single general cause of aneuploidies in cancer?

[0158] The demonstration that centromere breaks alone can account for the large majority of aneuploidies in cancer focuses attention on the possibility that merotelic attachments to a singleAttorney Docket No.1426.48.WO chromatid splits the centromere in two during anaphase (7). Centromeres are fragile sites in the genome (29, 31-34), but the amount of force required for breaking DNA (19) is vastly in excess of the 5-7 pN required to rupture a single kinetochore-DNA interaction (35). Although merotelic attachments of spindle microtubule bundles to a single kinetochore might in principle break centromeres at anaphase, chromatin is elastic (19), and single centromeres become distorted when pulled towards opposite poles, perhaps owing to the bipartite organization of the centromere (16, 20).

[0159] The patient-specific whole-arm loss bias that we observed across tumor types and in RNAPII, RNA-seq and WGS data points to a general cause of aneuploidy in cancer that similarly affects all chromosome arms in each patient. This possibility was envisioned by Boveri, who proposed that an “abnormal event” in the primordial cancer cell precedes aneuploidy (26). To explain the generation of whole-arm aneuploids, we have proposed that excess H3 histones resulting from overexpression of S- phase-dependent histone genes in cancer compete with CENP-A for incorporation at centromeres (6) (FIG. 25A). CENP-A is the histone H3 variant that marks active centromeres and is essential for kinetochore function (16). Depletion of CENP-A during S phase can lead to transcription-replication conflicts and R-loops that stall the replication machinery, resulting in fork collapse and chromosome breaks of the type that can lead to whole-arm aneuploids (36-38) and replacement with H3 nucleosomes (39, 40). As these S-phase events occur during a different phase of the cell cycle than mitotic errors, S-phase and mitosis models are mutually exclusive in that a centromere break could have occurred before a mitotic error or vice versa, but not at the same time. However, it is possible that mitotic segregation errors help to perpetuate whole-arm aneuploids once they are generated by a break. Although 21.6% of aneuploidies were scored as whole-chromosome aneuploids in TCGA data (10), breaks between functionally bipartite human centromeres with merotelic attachments to each half-centromere (16) would result in separated p and q arms, and simultaneous gain or loss might be mis-scored as whole-chromosome aneuploidies.

[0160] How might centromere breaks account for the excess of whole-arm losses over gains? Hypertranscription and over-expression of S-phase-dependent histones compete with CENP-A nucleosomes (40), resulting in centromere breaks (FIG. 25B). If a centromere break releases a chromosome arm that lags at anaphase, it may form a micronucleus upon mitotic exit (41), eventually to undergo degradation in the cytoplasm resulting in a whole-arm loss. However, in cases where S- phase replication occurs within a micronucleus and is followed by kinetochore formation on the broken centromere and monopolar microtubule capture, then the nucleus reforming at that pole duringAttorney Docket No.1426.48.WO the following telophase will include two extra chromosome arms. In this way, whole-arm gains would be secondary events, sometimes leading to replication but at other times to degradation and so would be less frequent than whole-arm losses, as we observed.

[0161] As whole-arm gains and losses are by far the dominant aneuploid type in cancer (12), prevention of centromere breaks could be a general therapeutic strategy. Preventing over-expression of histone genes might be such a strategy. S-phase-dependent histone genes have unique therapeutic vulnerabilities. These genes are present in clusters located in phase-separated histone locus bodies (HLBs) where they are the only RNAPII transcripts with 3’ stem-loops that are recognized by the stem-loop binding protein (42). S-phase-dependent 3’-end RNA processing within the HLB is mediated by the U7 small nuclear ribonucleoprotein complex without polyadenylation. Also, S-phase- dependent histone genes are uniquely activated for transcription by the NPAT protein, which is the major structural component of the HLB, and are uniquely repressed outside of S-phase by soluble histone H4 (43). Like RNAPII, which is the target of multiple general anti-cancer therapies, the S- phase-dependent system of gene regulation is ancestral for eukaryotes (44), and so anti-histone therapies may be less likely than targeted therapies to induce resistance. Thus, the generality of the whole-arm loss bias that was shown herein may open the way to development of entirely new general anti-cancer therapeutic options. Materials and Methods RNA-seq data processing and analysis

[0162] The RNA-seq data for 1,298 meningioma samples and corresponding recurrence clinical data were obtained from 13 meningioma studies compiled by Thirimanne et al. (8). Raw FASTQ files were re-aligned using STAR (version 2.7.11) to the hg19 reference genome from GENCODE (v19). Unstranded RNA-seq counts were used as raw counts per gene per sample.

[0163] Chromosome arm gains and losses were inferred using CaSpER (9), with the function extractLargeScaleEvents from the CaSpER R package. The default threshold of 0.75 was applied for calling large-scale chromosomal alterations. Kaplan-Meier survival curves were generated using the survminer R package, and log-rank tests were performed to evaluate the significance of the differences in survival curve distributions. Whole-genome sequencing data analysis

[0164] Whole-genome sequencing (WGS) data and associated survival information were obtained from The Cancer Genome Atlas (TCGA) (https: / / portal.gdc.cancer.gov). Whole chromosome armAttorney Docket No.1426.48.WO aneuploidy was determined using the ABSOLUTE algorithm (45), and results were directly retrieved from Table S2 of Taylor et al. (10)

[0165] To assess recurrence outcomes, we used disease-free interval (DFI) as the primary survival metric, as recommended (11). Statistical comparisons of whole chromosome arm aneuploidy between acrocentric and metacentric chromosomes were conducted using the Wilcoxon rank-sum test, with significance levels indicated by corresponding p-values in boxplot analyses.

[0166] The following references pertain to Example 3: 1. Hansemann D (1891) Ueber pathologische Mitosen. Virchows Arch Pathol Anat 123:356-370. 2. Gordon DJ, Resio B, & Pellman D (2012) Causes and consequences of aneuploidy in cancer. Nat Rev Genet 13:189-203. 3. Ben-David U & Amon A (2020) Context is everything: aneuploidy in cancer. Nat Rev Genet 21:44-62. 4. Deeg HJ, et al. (2012) Five-group cytogenetic risk classification, monosomal karyotype, and outcome after hematopoietic cell transplantation for MDS or acute leukemia evolving from MDS. Blood 120:1398-1408. 5. Henikoff S, et al. (2023) Epigenomic analysis of Formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun 14:5930. 6. Henikoff S, et al. (2025) RNA polymerase II at histone genes predicts outcome in human cancer. Science 387:737-743. 7. Martinez AC & van Wely KH (2010) Are aneuploidy and chromosome breakage caused by a CINgle mechanism? Cell Cycle 9:2275-2280. 8. Thirimanne HN, et al. (2024) Meningioma transcriptomic landscape demonstrates novel subtypes with regional associated biology and patient outcome. Cell genomics 4:100566. 9. Serin Harmanci A, Harmanci AO, & Zhou X (2020) CaSpER identifies and visualizes CNV events by integrative analysis of single-cell or bulk RNA-sequencing data. Nat Commun 11:89. 10. Taylor AM, et al. (2018) Genomic and Functional Approaches to Understanding Cancer Aneuploidy. Cancer Cell 33:676-689 e673. 11. Liu J, et al. (2018) An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics. Cell 173:400-416 e411. 12. Shih J, et al. (2023) Cancer aneuploidies are shaped primarily by effects on tumour fitness. Nature 619:793-800. 13. Oztek MA, et al. (2023) Changes to pediatric brain tumors in 2021 World Health Organization classification of tumors of the central nervous system. Pediatr Radiol 53:523-543. 14. Cimino PJ, et al. (2017) Multidimensional scaling of diffuse gliomas: application to the 2016 World Health Organization classification system with prognostically relevant molecular subtype discovery. Acta neuropathologica communications 5:39. 15. Logsdon GA, et al. (2024) The variation and evolution of complete human centromeres. Nature 629:136-145. 16. Sacristan C, et al. (2024) Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin. Cell 187:3006-3023 e3026.Attorney Docket No.1426.48.WO 17. Ghevaria H, et al. (2022) Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes. International journal of molecular sciences 23:10.3390 / ijms23020665. 18. Geyer J, et al. (2025) Real-time genomic characterization of pediatric acute leukemia using adaptive sampling. Leukemia 10.1038 / s41375-025-02565-y. 19. Bloom KS (2008) Beyond the code: the mechanical properties of DNA as they relate to mitosis. Chromosoma 117:103-110. 20. Darlington CD (1939) Misdivision and the genetics of the centromere. Journal of Genetics 37:361-374. 21. Feser J, et al. (2010) Elevated histone expression promotes life span extension. Mol Cell 39:724-735. 22. Hassold T & Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2:280-291. 23. Weaver BA & Cleveland DW (2008) The aneuploidy paradox in cell growth and tumorigenesis. Cancer Cell 14:431-433. 24. Duijf PH, Schultz N, & Benezra R (2013) Cancer cells preferentially lose small chromosomes. Int J Cancer 132:2316-2326. 25. Boveri T (1914) Zur Frage der Entstehung maligner Tumoren. Gustav Fischer Verlag, Jena, Germany. 26. Harris H (2008) Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Harris. Preface. J Cell Sci 121 Suppl 1:v-vi. 27. Duesberg P (2007) Chromosomal chaos and cancer. Sci Am 296:52-59. 28. Lynch A, Bradford S, & Burkard ME (2024) The reckoning of chromosomal instability: past, present, future. Chromosome Res 32:2. 29. Guerrero AA, et al. (2010) Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle. Proc Natl Acad Sci U S A 107:4159-4164. 30. Maiato H & Silva S (2023) Double-checking chromosome segregation. J Cell Biol 222. 31. Kolbin D, et al. (2025) Centromeres are stress-induced fragile sites. Curr Biol 35:1197- 1210 e1194. 32. Scelfo A, et al. (2024) Specialized replication mechanisms maintain genome stability at human centromeres. Mol Cell 84:1003-1020 e1010. 33. Scelfo A & Fachinetti D (2023) Centromere: A Trojan horse for genome stability. DNA Repair (Amst) 130:103569. 34. Lane KA, et al. (2025) PBRM1 directs PBAF to pericentromeres and protects centromere integrity. Nat Commun 16:1980. 35. Akiyoshi B, et al. (2010) Tension directly stabilizes reconstituted kinetochore- microtubule attachments. Nature 468:576-579. 36. Giunta S, et al. (2021) CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy. Proc Natl Acad Sci U S A 118. 37. Giunta S & Funabiki H (2017) Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T. Proc Natl Acad Sci U S A 114:1928-1933. 38. Talbert PB & Henikoff S (2022) The genetics and epigenetics of satellite centromeres. Genome Res 32:608-615. 39. Chen CC, et al. (2015) Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription. Dev Cell 34:73-84. 40. Blower MD, Sullivan BA, & Karpen GH (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2:319-330.Attorney Docket No.1426.48.WO 41. Cimini D (2023) Twenty years of merotelic kinetochore attachments: a historical perspective. Chromosome Res 31:18. 42. Geisler MS, Kemp JP, Jr., & Duronio RJ (2023) Histone locus bodies: a paradigm for how nuclear biomolecular condensates control cell cycle regulated gene expression. Nucleus 14:2293604. 43. Ahmad K, et al. (2024) Histone H4 limits transcription of the histone locus in Drosophila. biorxiv doi:10.1101 / 2024.12.23.630206. 44. Davila Lopez M & Samuelsson T (2008) Early evolution of histone mRNA 3' end processing. RNA 14:1-10. 45. Carter SL, et al. (2012) Absolute quantification of somatic DNA alterations in human cancer. Nat Biotechnol 30:413-421. Example 4. Cell-cycle-dependent repression of histone gene transcription by histone H4

[0167] In all eukaryotes DNA replication is coupled to histone synthesis to coordinate chromatin packaging of the genome. Canonical histone genes coalesce in the nucleus into the Histone Locus Body (HLB), where gene transcription and 3’ mRNA processing occurs. Both histone gene transcription and mRNA stability are reduced when DNA replication is inhibited, implying that the Histone Locus Body senses the rate of DNA synthesis. In Drosophila melanogaster, the S-phase- induced histone genes are repeated in an ~100 copy repeat unit array, whereas in humans, these histone genes are scattered. In both organisms these genes coalesce into Histone Locus Bodies. In this example, a transgenic histone gene reporter and RNAi in Drosophila was used to identify canonical H4 histone as a unique repressor of histone synthesis during the G2 phase in germline primary spermatocytes. Using cytology and CUT&Tag chromatin profiling, it was found that histone H4 uniquely occupies histone gene promoters in both Drosophila and human cells. The results suggest that repression of histone genes by soluble histone H4 is a conserved mechanism that coordinates DNA replication with histone synthesis in proliferating cells.

[0168] The genome of eukaryotic cells is packaged into nucleosomes, where DNA is wrapped around histone octamers. In animal cells, the genes encoding canonical histone proteins are highly distinctive: These multi-copy genes are abundantly transcribed by RNA Polymerase II (RNAPII) during S phase of the cell cycle, and are the only protein-coding genes that produce transcripts without introns or 3’ polyadenylation [Duronio & Marzluff 2017]. Histone genes nucleate a distinctive body within the nucleus termed the Histone Locus Body (HLB), where specific transcription factors and RNA processing proteins localize. The HLBs of Drosophila and mammals share fundamental molecular components, including the Cyclin E / CDK2-activated transcription cofactor Mxc / NPAT, 3’ mRNAAttorney Docket No.1426.48.WO stem-loops, Stem Loop Binding Protein (SLBP) and the U7 snRNP 3’-end processing machinery. Despite these cytological and compositional similarities, the histone genes are radically different in gene organization: In Drosophila melanogaster the five canonical histone genes (H1, H2A, H2B, H3 and H4), are arranged in a unit tandemly repeated ~100 times at one locus [Liu et al 2006], whereas in humans 72 genes are scattered with a major cluster on Chromosome 6 and two minor clusters on Chromosome 1. These human genes are non-repetitive and embedded in euchromatic regions of chromosomes, while the tandemly repeated Drosophila genes are subject to heterochromatic silencing [Elgin 1996], and it has been unclear how many of these 200 gene repeat units in a diploid cell are transcribed. By profiling both D. melanogaster and human histone genes, we aim to understand ancient conserved mechanisms of S-phase-dependent histone gene regulation that have endured despite profound genomic and epigenomic changes.

[0169] Here, Drosophila histone gene rescue constructs [Günesdogan et al 2010; MacKay et al 2015] and fluorescently marked histone transgene reporters [Shindo & Amodeo 2019] were utilized to test for histone gene derepression after knockdown of candidate repressors in the synchronized G2 phase gonial cells of testes. We discovered that reduction of histone H4 strongly derepressed histone gene expression outside of S phase, but no other candidate regulator had an effect. Using imaging, it was shown that histone H4 localizes to the HLB in Drosophila cells; using CUT&Tag chromatin profiling, histone H4 was precisely localized to histone gene promoters, coinciding with peaks of Mxc / NPAT, initiating RNAPII and of active chromatin. Turning to human K562 cells, similar cytological localization of histone H4 to the HLBs and coincident genomic localization of histone H4, NPAT, and RNAPII at active histone genes was observed. These results imply a direct mechanism whereby excess histone H4 in cells buffers histone gene transcription to coordinate chromatin packaging with DNA replication. Chromatin features at active and silenced histone genes in Drosophila

[0170] As the histone genes in the Histone Locus Body (HLB) are repetitive, and normal cells contain both active and silenced histone genes, mapping of chromatin features to a genome assembly cannot distinguish which features are associated with which expression state. To address this, chromatin features were profiled in two genotypes: wildtype, where some of the ~200 histone genes must be active while others are silenced, and the “12XWT” line, where the histone locus has been deleted and a construct carrying 12 copies of the histone repeat unit (HRU) rescues the flies [Crain et al 2024]. All copies of the histone genes were expected to be active in this second genotype. By comparingAttorney Docket No.1426.48.WO chromatin profiles between these two genotypes, the chromatin features of active and of silenced histone genes were inferred.

[0171] Wing imaginal discs from male larvae were dissected as a sample of proliferating cells, and subjected them to CUT&Tag profiling [Kaya-Okur et al 2019], generating >1 million reads mapped to the dm6 genome assembly for each sample (Table 4). Antibodies to the HLB-specific transcription cofactors Mxc [White et al 2011] and Mute [Bulchand et al 2010] were first used. As expected, these two factors are localized to only the histone locus in the genome (FIG.30A). Signal for both factors is broadly dispersed across the His3-His4 and His2A-His2B gene pairs of the 5 kb HRU, with peaks at the divergent promoters of each pair, and no signal over the adjacent His1 gene (FIG.30B). These results are consistent with previous chromatin mapping of Mxc in Drosophila embryos [Hodkinson et al 2024]. The divergent His4-His3 promoter region nucleates HLB formation [Salzler et al 2013], and these binding profiles are consistent with the idea that Mxc binds at these sites in the histone locus and nucleates HLB formation.

[0172] To assess transcriptional activity of the histone genes, multiple components and isoforms of RNA polymerase II (RNAPII) were profiled in larval wing disc samples. As expected, the RNAPII component unphosphorylated Rpb1, and phosphorylated initiating (RNAPII-S5p) and elongating (RNAPII-S2p) isoforms of Rpb1 mark the histone locus in wildtype cells (FIGS.30A-30B). In fact, the histone locus is the major site of Rpb1 and RNAPII-S5p signal across the genome (FIG. 30A), accounting for 1.8% and 2.5% of mapped reads, respectively, while only 0.2% of reads for the RNAPII-S2p isoform map to this locus. This is consistent with cytological description of enrichment for the unmodified and initiating forms of RNAPII at the HLB in other Drosophila cell types [Huang et al 2021, Kemp et al 2021, Cho et al 2022]. Note that the His3-His4 and His2A-His2B have strong peaks of RNAPII-S5p and RNAPII-S2p isoforms at their promoters and across their gene bodies, indicating high transcription of these genes. In contrast, His1 has only a low broad distribution of these polymerase isoforms across its length (FIG.30B), implying that it is expressed at lower levels.

[0173] Signal counts for Mxc, Mute, and RNAPII were then compared between wing disc samples from wildtype and from 12XWT flies. Per-gene counts were calculated to adjust for the different numbers of copies in the two strains (100 copies / genome in wildtype, 12 copies / genome in 12XWT). The per-copy sum counts of Mute and Mxc signal are higher in wildtype than in 12X flies, and proportional to the numbers of histone genes in these genotypes (FIG.31A). In contrast, signals for the RNAPII-S5p isoform are dramatically increased, and signal for the RNAPII-S2p isoform shows aAttorney Docket No.1426.48.WO slight gain. Thus, each histone gene in the 12XWT line must carry more RNAPII than in wildtype. This is consistent with the idea that some histone genes are active and others silenced in wildtype, or alternatively that each gene is active an intermediate level. In either case, all histone genes in the 12XWT strain appear to be more heavily transcribed, presumably to support cell proliferation. Heterochromatic histone marks at silenced HLB genes

[0174] To determine histone modifications associated with histone genes, five modifications associated with active gene expression [Barth & Imhof 2010] were profiled (FIG.31B). In wildtype samples, all three methylation states of the histone H3K4 residue (H3K4me1, H3K4me2, and H3K4me3) and acetylation at histone H3-K27 (H3K27ac) are enriched at the wildtype histone locus at levels comparable to surrounding active enhancers and genes. In contrast, there is little detectable trimethylation of the K36 residue of histone H3 (H3K36me3), consistent with the intronless structure of histone genes [Schwartz et al 2009]. Adjusting for copy number, there is a substantial per-gene copy gain only in the H3K4me3 mark in the 12XWT line (FIG.31A) and no or moderate gains in the H3K4me1 and H3K4me2 marks. These changes are consistent with the higher average transcription of histone genes in this genotype.

[0175] Five histone modifications typically associated with silencing across histone genes between the two Drosophila lines were then examined. Three modifications are strongly enriched at the histone genes in wildtype samples: mono- and di-methylation of histone H3 at lysine-9 (H3K9me1 and H3K9me2 [Ebert et al 2006]), and ubiquitinylation of histone H2A at lysine-118 (uH2A [Barbour et al 2020]) (FIG.31B). These first two marks imply that histone genes have a partially heterochromatic character likely due to the repeated genes, although the locus lacks tri-methylation of histone H3 at lysine-9 (H3K9me3 [Ebert et al 2006]) (FIG.31B). The presence of uH2A at histone genes suggests that these are sites of PRC1 activity, although the canonical trimethylation mark of histone H3 at lysine-27 (H3K27me3 [Cao et al 2002]) mark of Polycomb-silenced domains is absent.

[0176] Since any histone modification associated with histone gene silencing should be present in wildtype but absent in the 12XWT line, silencing histone modifications in this rescue line were profiled, and per-gene copy changes calculated (FIG. 31A). Previous results have implicated H3K9 methylation in histone gene silencing [Ner et al 2002; Ito et al 2012]. Indeed, the per-gene copy coverage of the H3K9me1 and H3K9me2 marks drop in the 12XWT strain compared to wildtype. In contrast, per-gene density of the uH2A modification is slightly increased. There is very little of the H3K9me3 or H3K27me3 marks in either the wildtype or 12XWT strains. These results support theAttorney Docket No.1426.48.WO idea that inactive histone genes are marked with mono- and demethylation of the H3K9 residue, where the wildtype histone locus is a mixture of transcriptionally active histone genes mixed with silenced histone genes. This is analogous to the functional organization of ribosomal RNA genes, where actively transcribed units are intermixed with silenced units [Grummt & Pikaard 2003]. In both cases a mixture of active and silenced genes may allow cells to fine-tune histone production to the needs of cell proliferation and growth, the rates of which vary between tissues and life stages. A visual reporter for histone gene silencing

[0177] Extra histone repeat unit transgenes are repressed in proportion to the number of total histone genes in a genotype [Chaubal et al 2023], an effect that appears similar to the reduced expression of histone genes in wildtype. To visualize histone gene expression in living animals, HRU reporter constructs were used where either the His3 or the His2A gene is fused to the octocoral Dendra2 fluorescent protein coding sequence [Shindo & Amodeo 2019; Methods]. These constructs express fluorescently tagged histones in eggs and developing embryos [Shindo & Amodeo 2019], and at low levels in proliferating cells of later stages such as in larval imaginal wing discs (FIG. 32A). It was reasoned that if these transgenes are partially repressed, then genetically interfering with histone gene silencing would produce more fluorescent protein. Indeed, the expression of the His2ADendra2 HRU transgene is dramatically increased ~37X in the 12XWT background compared to its expression in wildtype in wing imaginal discs (FIG.32B). This implies that cells with reduced histone gene numbers sense a dearth of histones, and upregulate all histone genes to compensate and provide for chromatin duplication.

[0178] While wishing to identify the mechanism by which histone genes are repressed, genetic reduction of such a factor might inhibit viability in the growing wing imaginal disc. Therefore, we turned to a non-essential tissue where we could still assess reporter expression with RNAi. The adult testis is dispensable for organismal viability. It also spatially organized so that the developmental and cell cycle stage of cells can be identified by their position in the tissue [White-Cooper 2010]. Germline stem cells are located at the apical testis tip and undergo four rounds of mitotic division in this proliferating zone, producing G2 phase spermatogonial cells. These then grow for ~4 days before meiosis and sperm differentiation. These stages were imaged by dissecting and fixing testes from males carrying a bamGAL4 driver with an inducible UAS-RFP transgene. This combination produces RFP specifically in gonial cells (FIG.33A). These testes were immunostained with antibodies to Mxc, a constitutive component of the HLB, and to phospho-Mxc, which is catalyzed by Cyclin E / Cdk2Attorney Docket No.1426.48.WO kinase in S phase of the cell cycle [White et al 2007]. The proliferating zone at the apical tip of the testis is marked with large HLBs containing phosphorylated Mxc, and abut RFP-stained G2 phase cells (FIG. 33A). Slightly more distal in the testis the Mxc-labeled HLB is divided into 2-4 smaller dots, consistent with the unpairing of homologous loci in this developmental stage [Vazquez et al 2002], and Mxc staining disappears from nuclei in later primary spermatocytes before meiosis.

[0179] Fluorescence from a control His2AVDendra2 histone variant gene (located on chromosome 3 outside of the histone locus) was imaged, and this variant protein is abundant throughout the apical tip of the testis (FIGS. 33B, 33C). In contrast, the HRU transgenes are repressed in the testis. No fluorescence from the His2ADendra2 transgene is apparent in the very apical tip including in bam- positive gonial cells, but then weakly appears in later stages (FIGS. 33D, 33E). Fluorescent protein from the His3Dendra2 HRU is undetectable throughout the testis, with signal only in the nuclei of somatic sheath cells (FIGS. 33F, 33G). The difference in pattern of expression between His2ADendra2 and His3Dendra2 transgenes was attributed to the deposition of histone H2A in post- mitotic cells, while histone H3 cannot [Jackson & Chalkley 1985, Kimura & Cook 2001]. This repression is sensitive to the demand for histones, because animals with reduced numbers of histone genes and the His2ADendra2 reporter HRU now show intense fluorescence throughout the apical tip of the testis, and gonial cells (FIG.33H). Thus, it was inferred that germline cells – like somatic cells – upregulate histone gene expression when these genes are limiting. Reduced histone H4 activates histone gene reporters

[0180] Previous work has implicated the H3K9-methyltransferase Suppressor of variegation 3-9 (Su(var)3-9) in histone gene silencing [Ner et al 2002; Ito et al 2012], and indeed we find that the histone locus is enriched for H3K9 methylation (FIG. 31B). We constructed viable null Su(var)3-9 flies from transheterozygous point mutation alleles [Schotta et al 2002],His2ADendra2 nor His3Dendra2 reporters are derepressed in this background (FIG.33I). To identify mechanisms responsible for HRU repression, we targeted a selection of chromatin proteins for RNAi knockdown in the gonial cell stage. None of these knockdowns derepressed His2ADendra2 or His3Dendra2 reporters, including the histone H3 lysine-9 methyltransferase eggless (egg [Clough et al 2007]), the H3K9me2 / 3-binding proteins HP1 [James et al 1989] or HP2 [Shaffer et al 2002], the histone H2A ubiquitin ligase Sex combs extra (Sce [Gutierrez et al 2012]), the PRC1 component Polycomb (Pc [Lanzuolo & Orlando 2012]), or the histone H3 lysine-27 methyltransferase Enhancer of zeste (E(z) [Cao & Zhang 2004]). Thus, it appears that while silenced HRUs carry modificationsAttorney Docket No.1426.48.WO associated with heterochromatic silencing, these modifications do not determine expression of the histone gene reporter; instead, the predominant mechanism of regulating the histone genes is in response to the demand for chromatin packaging in proliferating cells.

[0181] Since changes in histone gene number do alter reporter expression, RNAi was used to knockdown histones in gonial cells of the male germline. Knockdown of the linker histone gene His1, the core histone genes His2A, His2B, His3, and His4, of the histone variant genes His2AV, His3.3A, and His3.3B, as well as the orphan gene His4R were tested. Testes for all of these genotypes contain sperm and males are fertile, implying that these knockdowns only partially reduce histone gene expression. The HRU reporters remained repressed in 8 / 9 of these knockdowns. Only knockdown of the His4 gene resulted in dramatic derepression of both His2ADendra2 and His3Dendra2 in gonial cells (FIGS.33I-33M). This implies that cells measure the demand for histones based only on the H4 histone. Histone H4 localizes to the HLB in Drosophila cells

[0182] It is surprising that knockdown of only one histone modulates HRU silencing, since histones associate in dimers and in octamers as nucleosomes are assembled, although some examples where monomeric histone are complexed with protein chaperones have been identified [Apta-Smith et al 2018], and instances where a singular histone is used to measure chromatin in both Drosophila and in human cells [Shindo & Amodeo 2021; Kobiyama et al 2010]. To test if histone H4 localizes to the HLB on its own, we examined the localization of different histones within the male germline using inducible GFP-tagged constructs [Schwartz & Ahmad 2005; Methods]. Induction of a tagged H3 or tagged H3.3 histones in gonial cells broadly labels the nuclei of these cells (FIGS. 34A, 34B). In contrast, tagged H4 histone shows a distinct subnuclear pattern, with one major dot in each nucleus (FIG. 34C). This dot coincides with Mxc protein at the HLB in gonial cells (FIG. 34D). Since the bamGAL4-induced H4GFP construct is not expressed in early stages, testes were stained with an antibody to histone H4. This revealed an H4 dot in cells throughout the earlier proliferating zone, including most cells actively undergoing DNA replication (FIG.34E) and interphase cells (FIG.34F). Only 7% (1 / 14) of prophase cells show the dot (FIG.34G), consistent with the partial disassembly of the HLB in mitosis [White et al 2011]. Thus, this histone is in the right place to directly affect histone gene expression both in S phase and in gap phase cells.

[0183] Drosophila cultured Kc167 cells were examined to determine if histone H4 localizes to the HLB in other cell types. In immunostained samples, histone H4 staining colocalizes with Mxc at HLBsAttorney Docket No.1426.48.WO (FIG.34H). In chromatin profiling, histone H4 shows high signal only at the histone locus, coincident with Mxc, H3K27 acetylation, and RNAPII-S5p signal (FIG.34I). As Kc167 cells are derived from somatic embryonic cells, this implies that non-nucleosomal histone H4 is a general component of the HLB. Loss of histone H4 alters HLB activity

[0184] The HLB is enriched for the initiating RNAPII-S5p isoform in embryos and in the female germline [Kemp et al 2021, Cho et al 2022, Lu et al 2024]. In the testis, RNAPII-S5p is distributed through nuclei and forms a bright focal spot at the HLB in proliferating zone nuclei with bright phospho-Mxc staining, and slightly weaker focal staining in RFP-positive G2 phase gonial cells (FIGS.35A-35C). This is the major isoform of RNAPII engaged at histone genes, as staining for the RNAPII-S2p isoform is broadly distributed throughout nuclei with no greater enrichment at the HLB (FIG.35D). By the early primary spermatocyte stage the HLB is no longer enriched for any RNAPII isoform. We infer that active histone loci in the proliferating zone are engaged with high levels of the RNAPII-S5p isoform, and a reduced amount of this isoform persists in G2 gonial phase cells when histone genes are no longer expressed. Overall, the HLB in the testis progresses from having high levels of engaged RNAPII in the proliferating zone, to less engaged and non-transcribing RNAPII in G2 phase gonial cells, and to loss of RNAPII in primary spermatocytes. Dissolution of the HLB occurs in later stages, as Mxc staining is eventually lost, as cells do not need histone gene expression as they proceed to meiosis and sperm differentiation. This progression and the spatial arrangement of the testis is an easily tractable setting to follow changes in the HLB.

[0185] The bamGAL4-induced H4-GFP dot is intense in G2 phase gonial cells, and this coincides with reduced RNAPII-S5p staining in G2 gonial cells (FIGS.36A, 36B), consistent with a role in limiting histone gene expression. We then examined testes where His4 was knocked down in these cells. In contrast to wildtype controls (FIGS. 36C, 36E), knock-down testes have elevated intensity of the RNAPII-S5p isoform in G2 gonial cells and increased staining of phospho-Mxc at HLBs (FIGS.36D, 36F). These defects implicate histone H4 in the switch from active to inactive forms of the HLB. Histone H4 localizes to active histone gene promoters in human cells

[0186] Since histone genes are repeated and we cannot distinguish between gene copies, we cannot define whether histone H4 binds all histone genes or only active or silent genes in Drosophila. But histone H4 is an ancient protein, and if it plays a role in limiting histone gene expression we expect HLB localization to be conserved across species. In the human genome the multiple copies of histoneAttorney Docket No.1426.48.WO genes are not in a repeat array; instead they are comparatively separated and scattered across four clusters, termed HIST1-4 [Dhahri et al 2024], and these aggregate into HLBs containing the transcription cofactor NPAT, the human homolog of Mxc [Ma et al 2000; Ghule et al 2008]. Indeed, immunostaining of human K562 cells reveals that NPAT and histone H4 colocalize at HLBs (FIG. 37A). We then profiled the distribution of histone H4 and the active histone modification H3K27 acetylation in K562 cells and compared these to previously published profiling of NPAT [Kaya-Okur et al 2019] and RNAPII-S5p [Janssens et al 2021]. As expected, 64 canonical histone genes in the HIST on chromosome 6 are marked with both RNAPII and with H3K27mac modifications, identifying them as active genes (FIGS.37B-37D). Each of these active histone genes are also marked with NPAT and histone H4 (FIG.37B), and at high resolution these four chromatin features coincide at promoters (FIG.37C). In contrast, the 8 non-transcribed canonical histone genes lack both NPAT and histone H4 (FIGS.37B, 37D), as do the 5 active histone variant genes that are not S phase-regulated (FIG. 37D). Actively transcribed non-histone genes neighboring HIST clusters also lack NPAT and histone H4 signal (FIG.37B). These results implicate histone H4 in the S phase regulation of canonical histone gene promoters in human cells. Table 4. mapped Sample ID species genotype antibody epitope readsAttorney Docket No.1426.48.WO H3K36m BT1942 D.melanogaster 12XWT 283 e3 12,842,116Attorney Docket No.1426.48.WO H3K4me BT3072 D.melanogaster w 281 1 5,498,442 H3K4me 5Attorney Docket No.1426.48.WO y_0320.DTma rked SH Hs K5xliDiscussion

[0187] The invention of eukaryotic chromatin required coordination of histone protein synthesis with DNA replication in S phase cells. DNA replication and histone synthesis must be coupled because even small imbalances are detrimental, given the large amounts of chromatin duplicated each S phase. Underproduction of histones will result in incomplete chromatin packaging, and this leads to exposure and damage of new DNA [Dreyer et al 2024]. Histone overproduction results in chromosome lossAttorney Docket No.1426.48.WO [Meeks-Wagner & Hartwell 1986] and is cytotoxic [Gunjan & Verreault 2003]. Feedback between these two processes provides just enough histones to package newly replicated DNA [Weintraub 1972; Duronio & Marzluff 2017]. Feedback control implies that S phase cells measure both ongoing DNA replication and the need for more histones. Histone production is modulated through two main controls: CDK2-catalyzed phosphorylation of Mxc / NPAT induce transcription canonical histone genes when cells commit to S phase [Armstrong & Spencer 2023], and cell cycle-regulated associations between histone mRNA processing factors stabilize transcripts in S phase [Marzluff et al 2008]; both these processes take place in HLBs [Romeo & Schümperli 2016]. S phase-induced histone mRNAs are the only protein coding transcripts that are not poly-adenylated in animals; instead, these transcripts have a terminal stem-loop structure that is bound by stem-loop binding protein (SLBP), directing 3’ processing [Marzluff et al 2008]. Mathematical modeling suggested that feedback from soluble histone pools is necessary for precise coupling between DNA replication and histone synthesis [Christopher et al 2016]. The observations suggest a simple model where soluble histone H4 protein directly represses histone gene transcription. Ongoing DNA replication and chromatin packaging use up soluble histones, but once DNA replication ceases, soluble histones including histone H4 accumulate. Soluble histone H4 then localizes to the HLB and represses transcription. Although histone H4 localizes to histone gene promoters, repression might occur by modulating transcription or 3’ processing since both processes occur within the HLB.

[0188] The atypical gene structure appears to be the ancestral organization of S phase-induced histone genes throughout Eukaryota, because both stem-loop mRNA structures and SLBP homologs have been identified in protozoa at the base of the eukaryotic tree [Lopez & Samuelsson 2008]. The evolutionary origin of this system was unclear, but recently similarities to 3’ processing of transcripts in bacteria were pointed out [Shine et al 2024]. Thus, the histone 3’ processing system appears to be one of the few relics in eukaryotic genomes of their origin and may explain why these genes are sequestered in their own nuclear body. The eukaryotic histones themselves were derived from bacterial or viral proteins in the last eukaryotic common ancestor [Talbert et al 2022], with eukaryotic histone H4 being a sister lineage to both the HMfB archaeal histones and the doublet H4-H3 histones of the Nucleocytoviricota giant viruses [Grau-Bové et al 2022], that later diversified into the four core histone subtypes [Malik and Henikoff 2003]. Thus, it is conceivable that histone H4 has been used as a negative regulator of core histone gene expression all this time. Indeed, histone H4 is distinctive in that variants for this isotype rarely occur across eukaryotic evolution. One exception is a variantAttorney Docket No.1426.48.WO histone H4 encoded by some symbiotic bracoviruses; braconid wasps harboring this virus inject viral DNA into host moth larvae, where production of the variant histone suppresses host histone H4 mRNA production [Gad & Kim 2009]. This unusual variant appears to have weaponized the normal negative feedback loop of histone H4 on histone gene regulation for parasite life history.

[0189] Histone gene regulation has been implicated in cancer progression in human patients, and histone overproduction is predictive of cancer malignancy [Henikoff et al 2025]. There are multiple theories to explain the initiation of a cancer, invoking genetic mutation, changes in epigenetic marks, and defects in developmental signaling. Although the relative importance of these theories is now debated [Jassim et al 2023; Huang et al 2025], in any scenario progenitor cells must maintain a relatively undifferentiated state and proliferate. We have described that widespread chromosome arm aneuploidies are common in cancers and number of arm losses scales with malignancy [Zheng et al 2025]. Such aneuploidies in tumors were suggested to inhibit cellular differentiation and thereby trap anaplastic cells in a proliferative stage [Hansemann 1890; reviewed in Henikoff & Ahmad 2025]. We have proposed that these events are causally linked: that histone overexpression during S phase compromises histone variant-based centromere assembly and also accelerates cell proliferation; these effects would directly cause mitotic chromosome errors and result in aneuploidies [Zheng et al 2025; Henikoff & Ahmad 2025]. In support of this scenario, a very recent study identified reduced cell cycle duration as the only common feature of multiple distinct cancers, showing that tumorigenesis could be blocked by mutations affecting CDK2 activity [Chen et al 2025]. CDK2 is the kinase that phosphorylates Mxc / NPAT and histone gene activation, linking conserved HLB regulation described here to a deeper understanding of cancer. As such, detailing the mechanisms by which histone synthesis is normally restrained should provide insights into originating oncogenic events and opportunities for intervention. Methods Fly strains

[0190] All crosses were performed at 25˚C. All mutations and chromosomal rearrangements used here are described in Flybase (http: / / www.flybase.org). The w1118strain was used as a wildtype control. The 12XWT strain is w ; DHisC; 12XWT [MacKay et al 2015]. The HRU reporter His3Dendra2 was previously described [Shindo & Amodeo 2019], and the His2ADendra2 reporter was constructed similarly. Inducible histone lines were UAS-H3-GFP [Schwartz & Ahmad 2005], UAS-H3.3-GFP [Schwartz & Ahmad 2005], and the UAS-H4-GFP construct [Ahmad & Henikoff 2002] was injectedAttorney Docket No.1426.48.WO into fly embryos for P element transformation [Rubin & Spradling 1982] by BestGene Inc. (Chino Hills CA). A similar UAS-H4-eGFP construct used here for some experiments was previously published [Wooten et al 2019]. Additional constructs used for cytological characterization were y w P[bam-GAL4:VP16,w+]1 / Y ; P[UAS-RFP,w+]2. Inducible knockdown constructs and Bloomington Drosophila Stock Center IDs for histones and chromatin regulators are listed in Table 5. Antibodies

[0191] Antibodies used for CUT&Tag profiling and for immunocytology are listed in Table 5. Imaging fresh tissues

[0192] Dissected tissues from larvae or adults were mounted in PBS on slide and imaged by epifluorescence on an EVOS FL Auto 2 inverted microscope (Thermo Fisher Scientific) with a 10X, 20X, or 40X objective. Pseudo-colored images were adjusted and composited in Adobe Photoshop and Adobe Illustrator. Imaging immunostained testes

[0193] Testes from one-day old adult males were dissected in PBS, incubated in AccutaseTM(Stemcell Technologies, #07920) for 10 minutes at room temperature to permeabilize the tissue, fixed in 4% formaldehyde / PBS with 0.1% Triton-X100 (PBST) for 10 minutes, incubated twice in 0.3% sodium deoxycholate / PBST for 10 minutes each [Lim & Fuller 2012], incubated with primary antibodies in A+t buffer at 4˚C overnight, and then with fluorescently-labeled secondary antibodies (1:200 dilution, Jackson ImmunoResearch). Testes were stained with 0.5 μg / mL DAPI / PBS and mounted in 80% glycerol on slides, and imaged on a Stellaris 8 confocal microscope (Leica) with 20X or 63X objectives. Pseudo-colored images were adjusted and composited in ImageJ, Adobe Photoshop, and Adobe Illustrator. Imaging tissue culture cells

[0194] Drosophila Kc167 and human K562 cells were swelled with a hypotonic 0.5% sodium citrate solution, then smashed onto glass slides in a Cytospin 4 centrifuge (Thermo). Slides were fixed with 4% formaldehyde / PBST and incubated with primary antisera in A+t buffer, then with fluorescently- labeled secondary antibodies (1:200 dilution, Jackson ImmunoResearch), stained with 0.5 μg / mL DAPI / PBS, mounted in 80% glycerol, and imaged by epifluorescence on an EVOS FL Auto 2 inverted microscope (Thermo Fisher Scientific) with a 40X objective. Pseudo-colored images were adjusted and composited in Adobe Photoshop and Adobe Illustrator. CUT&Tag chromatin profilingAttorney Docket No.1426.48.WO

[0195] To perform CUT&Tag [Kaya-Okur et al 2019], we dissected 20 imaginal wing discs from male 3rd instar larvae in PBS buffer, and transferred them to a tube containing Accutase (Stemcell Technologies, #07920) at 25˚C for 30 minutes. We then added an equal volume of 30% BSA to block proteases, and ran the material through a 30 ½ gauge needle once to dissociate tissue. Tissue suspensions were divided between 4-8 reaction tubes, and bound with BioMag Plus ConA (Bangs Inc., #531) magnetic beads. Tissue culture samples in media were added directly to ConA beads for binding, and then lightly fixed onto beads with 0.1% formaldehyde at room temperature for 1’. All samples were incubated with the following CUT&Tag solutions sequentially: primary antibodies diluted in Wash+ buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% triton-X100, 2 mM EDTA, 1% BSA, with Roche cOmplete protease inhibitor) overnight at 4˚, secondary antibodies (in Wash+ buffer) for 1 hour at room temperature, and then incubated with pAGTn5 (Epicypher 15-1017) in 300Wash+ buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.05% triton-X100 with cOmplete protease inhibitor) for 1 hour. After one wash with 300Wash+ buffer, samples were incubated in 300Wash+ buffer supplemented with 10 mM MgCl2 for 1 hour at 25˚C to tagment chromatin. Tissue culture samples were tagmented in CUTAC buffer (10 mM TAPS pH 8.5, 20% DMF) supplemented with 5 mM MgCl2to enhance tagmentation efficiency. Samples were washed with 10 mM TAPS pH 8.5 and DNA was released with 0.1% SDS, 0.012 U / μL thermolabile protease K (NEB P8111S) in 10 mM TAPS at 37˚ and inactivated at 55˚C. Libraries were enriched with 14 cycles of PCR as described [Kaya-Okur et al 2019], and sequenced in dual-indexed paired-end mode (PE50) on the Illumina NextSeq 2000 platform at the Fred Hutchinson Cancer Center Genomics Shared Resource. Paired-end reads were mapped to this assembly using Bowtie2 using parameters, e.g.: --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. Gene score tables

[0196] To summarize the enrichment of profiling features across histones, mapped reads were counted from the start to the end of each gene in .bam files using subreads / feature_counts with option ‘_o’. Counts for each replication-coupled histone were summed, and counts for all genes were scaled by total mapped reads to give Counts per Million (CPM) reads. Genomic display

[0197] Files of mapped reads were converted to genome coverage with bedtools / bamcoverage [Quinlan & Hall 2010] and displayed in the UCSC genome browser [Kent et al 2002]. Selected regions were exported as PDFs and formatted with Adobe Illustrator.Attorney Docket No.1426.48.WO Data availability

[0198] Sequencing data have been deposited in GEO under accession code GSE280833. Data for NPAT profiling in K562 cells (SH_Hs_NPA1_20190217, SH_Hs_NPA2_20190217, SH_Hs_NPA4_20190217, SH_Hs_NPA8_20190217, SH_Hs_NPB1_20190217, SH_Hs_NPB2_20190217, SH_Hs_NPB4_20190217, and SH_Hs_NPB8_20190217 was previously published in Kaya-Okur et al 2019, and were merged here into one file (SH_Hs_NPB4_20190217). Data for RNAPII-S5p profiling in K562 cells (SH_Hs_K5xlin_PolS5P_3cy_0320, SH_Hs_K5xlin_PolS5P_6cy, SH_Hs_K5xlin_PolS5P_9cy_0320, SH_Hs_K5xlin_PolS5P_12cy_0320, and SH_Hs_K5xlin_PolS5P_20k_0320 were previously published in Janssens et al 2021 and were merged here in one file (SH_Hs_K5xlin_PolS5P_20k_0320). Table 5. Key Resources Table Reagent type Designation Source Identifiers Additional info ag) ag)ag)ag)ag)ag)ag)ag)ag)ag)ag)ag)ag)ag)ag)Attorney Docket No.1426.48.WO anti-rabbit IgG (guinea pig)Antibodies Online ABIN101961(1:100 CUT&Tag)anti-mouse IgG (rabbit) Abcam ab46540 (1:100 CUT&Tag)g)15 15do ntnt, eoAttorney Docket No.1426.48.WO Su(var)3-92 BDSC 6210GD-27042 (His1 RNAi) VDRC 27042 Dietzl et al 2007Attorney Docket No.1426.48.WO P[TRiP.HMS00066]attP2 (E(z) RNAi) BDSC 3365919Example 5. Protocol Development

[0199] An example protocol that can be used in the disclosure is described below, with an example overview in FIGS.38A-38C. Purify RNA

[0200] Harvest cells

[0201] Assess cell number and viability

[0202] Collect desired number and wash with 1x PBS by centrifugation for 5 m at 300 rcf.

[0203] Remove supernatant and flash freeze pellet in liquid nitrogen.Attorney Docket No.1426.48.WO

[0204] Store at -70C until ready to extract.

[0205] Purify RNA and genomic DNA

[0206] Follow the Macherey-Nagel NucleoSpin® RNA / DNA buffer kit combined protocols precisely to purify total RNA and genomic DNA from pelleted and frozen cells of interest. Genomic DNA should be stored at -20C, RNase inhibitors should be added to total RNA (1unit of inhibitor per microliter of solution) and the mixture kept at -20C short-term or -70 °C for longer.

[0207] Critical Note: After extraction, RNA should be kept on ice or frozen. RNA Quality Check

[0208] Concentration and purity ratios can be determined using a spectrophotometer.

[0209] Concentration: use the A260 to calculate RNA concentration (RNA concentration (μg / mL) = A260 * 40 * Dilution Factor)

[0210] Purity ratios: A260 / 280 ~ 1.9–2.1, A260 / 230 ~ 2.0 – 2.2

[0211] RNA Integrity via Agilent TapeStation (or other fragment analyzer) – RNA Integrity Number equivalent (RINe)

[0212] Follow the instructions for analysis of RNA on the High Sensitivity RNA ScreenTape Analysis available at agilent.com, TapeStation RNA ScreenTape & Reagents.

[0213] Total RNA may need to be diluted to meet the detection parameters of the RNA tape.

[0214] RINewill provide an assessment of RNA degradation, RIN scores between 8 and 10 are considered to indicate highly intact RNA.

[0215] Alternative methods for determining RNA integrity include agarose gel electrophoresis and visualization of ribosomal RNA.

[0216] Keep all components on ice throughout unless otherwise stated.

[0217] Synthesize cDNA Prepare reverse transcription reactions using Random Hexamers or oligo dT primers. Total RNA input should be 1μg for each reaction and should be prepared according to the M-MLV Reverse Transcriptase product literature.

[0218] Add the following components to a nuclease-free microcentrifuge tube: Table 6. Component Volume, μLAttorney Docket No.1426.48.WO 10 mM dNTP Mix (10 mM each) 1

[0219] Heat mixture to 65°C for 5 minutes [0220

[0221] Add to each of the above in order, do not master mix these. Table 7. Component Volume, μL 5X First-Strand Buffer 4

[0222] M, s.

[0223] Add 1 μL (200 units) of M-MLV RT, then mix by pipetting gently up and down.

[0224] Incubate tube for 10 minutes at 25°C (Random hexamer specific but ok for either preparation).

[0225] Incubate at 37°C for 50 minutes.

[0226] Heat to 70°C for 15 minutes to inactivate the enzyme.

[0227] Remove RNA by addition of 0.5 μL of RNase H, 5,000 U / mL, and incubate for 20 minutes at 37°C.

[0228] Freeze cDNA at -20C until ready to use. qPCR

[0229] Note: An 8-channel pipette aligns with every other well in a 384-well plate and allows for transfer from a 96-well format.

[0230] Dilute cDNA

[0231] Production of cDNA is assumed to produce the same as input RNA, ~ 1000 ng, which in a 20uL reaction gives a final concentration of 50ng / μL; input for each qPCR reaction should be 1ng.

[0232] Dilute an appropriate amount of cDNA to 0.5ng / uL intending to use 2uL per qPCR reaction.

[0233] Array the diluted cDNA into either a 96-well plate or 8-well strip tubes such that transfer to the final 384 plate is easily facilitated.

[0234] Note: To prevent cross-contamination, it is not recommended to keep cDNA from different samples on the same plate unless sufficient space is allowed to prevent carryover. Alternatively, they can be kept in separate plates or in individual strip-tubes.

[0235] Prepare and array Master MixesAttorney Docket No.1426.48.WO

[0236] Each 384-well qPCR plate will contain reactions for all samples prepared with either RH or OdT primer, two targets (histone primer pairs) and all housekeeping genes.

[0237] Samples and targets should be arrayed such that each paring has 4 replicates.

[0238] Prepare Master Mixes (MMs) containing SybrGreen PCR Mix and primers at a volume sufficient for quadruplicate reactions of each target / sample set; increase that volume ~10-20% for pipetting ease. Table 8. Reagent 1x rxn 2 S b ® G 10

[0239] Aliquot MMs into a row or two of a 96 well plates or 8-well strip-tubes, then use an 8-channel pipette to stamp into alternate wells of a 384-well plate, shifting as needed to array the reagents.

[0240] Add Template

[0241] Using an 8-channel pipettor, add 2uL of diluted cDNA into the appropriate well of 384 well plate with appropriate MM.

[0242] Pipette to mix.

[0243] Cover plate with optical film and seal well with plate film sealing tool

[0244] Spin plate to collect volume

[0245] Tap plate sharply against palm of hand on each side to thoroughly mix

[0246] Spin again to collect volume

[0247] Cycle as follows: 50C 2m, 95C 5m, 40x [95C 15s and 58C 30s], 40C 1m, followed by a melt curve 95C 1m, 60C 1m.

[0248] Once complete, collect plate for later

[0249] To collect data, select the analyze button and export resulting data to an Excel file Post-qPCRAttorney Docket No.1426.48.WO

[0250] Product analysis: Transfer 2uL of PCR product to 18uL of NF-water, mix well and use 2uL of dilution to determine fragment size using HSD1000 screentape in the 4200 TapeStation.

[0251] Amplicon Verification: PCR product was purified through a NucleoSpin clean-up column and quantified by spectrophotometry and submitted for Premium PCR sequencing at Plasmidsaurus which provides ~5K read depth of a mixed population sample and a consensus sequence.

[0252] Data analysis: Relative quantification is performed using ΔΔCT Method Stepwise Reagent, Consumable & Equipment List:

[0253] RNA Extraction Beckman Coulter ViCell BLU cell analyzer Macherey-Nagel NucleoSpin RNA, Mini kit for RNA purification, cat#740995.50 Macherey-Nagel NucleoSpin RNA / DNA Buffer Set for parallel RNA and DNA purification, cat# 740944 1M DTT – in house preparation Rnasin®Rnase Inhibitor (40U / uL), Promega N2515 RNase H, 5,000 U / mL, NEB #M0297

[0254] cDNA Synthesis M-MLV Reverse Transcriptase Kit (200 U / μL) ThermoScientific 28025013 Random Hexamers Primers (50 uM), Life Technologies N8080127 Oligo-dT 12-18 primers (0.5 ug / ul), Invitrogen 18-418-012 10 mM dNTP Mix (10 mM each), Kapa Biosystems KN1011 Rnasin®Rnase Inhibitor (40U / uL), Promega N2515

[0255] qPCR SYBR™ Green PCR Master Mix (2X), Applied Biosystems, Fisher Scientific 4309155 / 4364344 GSPrimers (10uM) – IDT, salt-free purification MicroAmp™ EnduraPlate™ Optical 384-Well Clear Reaction Plates, Thermo A36931 Optical covers: MicroAmp™ Optical Adhesive Film, Thermo 4360954 Macherey-Nagel NucleoSpin Gel and PCR Clean-up 740609.250

[0256] GSPrimers – IDT, salt-free, diluted to 10uM H1-5p 5'-AACAGCCGCATCAAGCT-3' (SEQ ID NO:2) H1-3p 5'-CCTTCTTGTTGAGYTTRAAGGA-3' (SEQ ID NO:3) H2A-5p 5'-GAGCTGGCNGGCAA-3' (SEQ ID NO:5)Attorney Docket No.1426.48.WO H2A-3p 5'-GGCARRACRCCRCCCT-3' (SEQ ID NO:6) H2B-5p 5'-CTGCCCGCCTGGC-3' (SEQ ID NO:10) H2B-3p 5'-TACTTGGTGACGGCCTT-3' (SEQ ID NO:11) H4-5p 5'-TCATCTACGAGGAGACYCG-3' (SEQ ID NO:17) H4-3p 5'-GCGYTTGGCGTGCTC-3' (SEQ ID NO:18)

[0257] Controls / primers 5'- GTCTCCTCTGACTTCAACAGCG-3' (SEQ I hGAPDH_F NO:21) 5'-ACCACCCTGTTGCTGTAGCCAA-3' (SEQ I hGAPDH_R NO:22) 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID ACTB-Forward Primer NO:23) 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID ACTB-Reverse Primer NO:24)

[0258] Equipment Nanodrop One Spec – Thermo Agilent TapeStation HSRNA tape (5067-5579) & reagents (5067-5580, 5067-5581) Centrifuge with plate rotor Eppendorf Mastercycler X50 Thermal cycler 384-well ABI QuantStudio™ 5 Real-Time PCR System running QuantStudio™ _Design and Analysis Software V1.4.3

[0259] An example standard curve with a serial dilution Ct plot is provided in FIG.39. The standard curve ensures that your primers bind to and amplify their target precisely and efficiently. qPCR efficiency by primer and cell line can be charted, with the slope of the regression line used to calculate the amplification efficiency (FIG.40). The target range is 90-110% efficiency, where at 100% one is doubling the amount of product in each cycle of the PCR reaction. Regarding quality control, melt curves highlighting K562 Wells at log10 (conc)= -2 are shown in FIG. 41, left. Melt curves with a single peak show specific product, with multiple peaks indicative of non-specific products. Absence of primer-dimers can be confirmed via melt curve or gel electrophoresis. Tapestation can be utilized to verify single product (FIG.41, right) and Sanger sequence confirmed, with results identifying whatAttorney Docket No.1426.48.WO was intended on histone proteins (FIG.42). Example data showing results of assay indicating relative abundance of targets in cancer cells, leukemia (top), myeloma (bottom) versus normal cells (light gray) (FIG.43).

[0260] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

1. Attorney Docket No.1426.48.WO THAT WHICH IS CLAIMED:

1. A method of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject, comprising detecting histone gene expression at one or more replication-coupled histone genes in a biological sample from the subject and comparing the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of histone gene expression at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of increased cancer aggressiveness and / or risk of cancer recurrence.

2. The method of claim 1, wherein detecting histone gene expression comprises subjecting the sample to a nucleic acid amplification (e.g., qRT-PCR).

3. The method of claim 1, wherein detecting histone gene expression comprises detecting RNA polymerase II (RNAPII) levels at one or more replication-coupled histone genes in the biological sample from the subject; and comparing the levels of RNAPII at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of RNAPII at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of increased cancer aggressiveness and / or risk of cancer recurrence.

4. The method of claim 3, wherein detecting RNAPII levels comprises subjecting the sample to Cleavage Under Targeted Accessible Chromatin (CUTAC) with an affinity agent comprising an antibody to a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.Attorney Docket No.1426.48.WO 5. A method of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject, comprising detecting chromatin accessibility at one or more replication-coupled histone genes in a biological sample from the subject and comparing the chromatin accessibility at the one or more replication-coupled histone genes in the biological sample to a reference, wherein an increase in the levels of chromatin accessibility at the one or more replication-coupled histone genes in the biological sample compared to the reference is indicative of increased cancer aggressiveness and / or risk of cancer recurrence.

6. The method of claim 5, wherein detecting chromatin accessibility comprises performing CUTAC.

7. The method of any one of claims 1-6, wherein the one or more of histone genes comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 of HIST2H3D, HIST2H4B, HIST2H3A, HIST2H2AA4, HIST2H2BC, HIST2H2AA4, HIST2H3 A, HIST2H4B, HIST2H2AC, HIST2H2AB, HIST1H2AA, HIST1H2BA, HIST1H2APS1, HIST1H1A, HIST1H3A, HIST1H4A, HIST1H4B, HIST1H3B, HIST1H2AB, HIST1H2BB, HIST1H3C, HIST1H1C, HIST1H4C, HIST1H1T, HIST1H2BC, HIST1H2AC, HIST1H1E, HIST1H2BD, HIST1H2BE, HIST1H4D, HIST1H2AD, HIST1H2BF, HIST1H4E, HIST1H2BG, HIST1H2AE, HIST1H3E, HIST1H1D, HIST1H4F, HIST1H4G, HIST1H3F, HIST1H2BH, HIST1H3G, HIST1H2BI, HIST1H4H, HIST1H2BJ, HIST1H2AG, HIST1H4I, HIST1H2AH, HIST1H2BL, HIST1H2AI, HIST1H3H, HIST1H2AJ, HIST1H2BM, HIST1H4J, HIST1H4K, HIST1H2AK, HIST1H2BN, HIST1H2AL, HIST1H1B, HIST1H3I, HIST1H4L, HIST1H3J, HIST1H2AM, HIST1H2BO, or any combination thereof.Attorney Docket No.1426.48.WO 8. The method of claim 7, wherein the one or more histone genes comprises one or more of gene Reference Sequences: NM_001123375, NM_001034077, NM_001005464, NM_001040874, NR_036461, NM_001040874 _ chr1_149822627, NM_001005464_ chr1_149824180, NM_001034077_ chr1_149832329, NM_003517, NM_175065, NM_170745, NM_170610, NR_045125, NM_005325, NM_003529, NM_003538, NM_003544, NM_003537, NM_003513, NM_021062, NM_003531, NM_005319, NM_003542, NM_005323, NM_003526, NM_003512, NM_005321, NM_021063, NM_003523, NM_003539, NM_021065, NM_003522, NM_003545, NM_003518, NM_021052, NM_003532, NM_005320, NM_003540, NM_003547, NM_021018, NM_003524, NM_003534, NM_003525, NM_003543, NM_021058, NM_021064, NM_003495, NM_080596, NM_003519, NM_003509, NM_003536, NM_021066, NM_003521, NM_021968, NM_003541, NM_003510, NM_003520, NM_003511, NM_005322, NM_003533, NM_003546, NM_003535, NM_003514, NM_003527, or any combination thereof.

9. A method of predicting cancer aggressiveness and / or risk of cancer recurrence in a subject, comprising detecting aneuploids in a biological sample from the subject and comparing the aneuploids in the biological sample to a reference, wherein an increase in the levels of aneuploids in the biological sample compared to the reference is indicative of cancer aggressiveness and / or risk of cancer recurrence.

10. The method of claim 9, wherein the increase in the levels of aneuploids are whole-arm chromosome losses.

11. The method of any one of the previous claims, wherein the reference is from non- cancerous tissue.

12. The method of any one of the previous claims, wherein the reference is a threshold value.Attorney Docket No.1426.48.WO 13. The method of any one of the previous claims, wherein the reference is based on a population of subjects.

14. The method of any one of the previous claims, wherein the reference is normal tissue from the subject.

15. The method of any one of the previous claims, further comprising contacting the sample with a known amount of spike-in DNA configured to facilitate calibration.

16. The method of any one of the previous claims, wherein the sample comprises tissue, blood, or cell-free DNA.

17. The method of any one of the previous claims, wherein the sample is from a tumor.

18. The method of any one of the previous claims, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

19. The method of any one of the previous claims, wherein the sample is from a subject with brain, breast, colon, liver, lung, stomach, kidney or rectum cancer.

20. The method of claim 19, wherein the brain cancer is meningioma.

21. The method of any one of the previous claims, further comprising performing a chromatin assay on a biological sample from the subject.Attorney Docket No.1426.48.WO 22. The method of any one of the previous claims, further comprising performing a chromatin accessibility assay on a biological sample from the subject.

23. The method of claim 22, wherein the chromatin assay is selected from a Cleavage Under Targeted Accessible Chromatin (CUTAC) assay, an Assay for Transposase-Accessible Chromatin (ATAC)-seq assay, a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-seq assay, a Nicking Enzyme-Assisted Accessible Chromatin Sequencing (NicE)-Seq assay, and a Reverse Transcribe and Tagment (RT&Tag) assay.

24. The method of any one of claims 21-23, comprising performing CUTAC wherein an affinity agent comprises an antibody to a phosphoform of the C-terminal domain of RNAPII, such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.

25. The method of any one of the previous claims, further comprising detecting RNAPII at one or more enhancers.

26. The method of any one of the previous claims, wherein the method is performed on a solid support.

27. The method of claim 26, wherein the solid support is a bead, a slide, or a well (e.g., a microwell or nanowell).

28. The method of any one of the previous claims, wherein the subject had cancer and the method further comprises administering to the subject a prophylactic treatment to the subject identified at increased risk of cancer recurrence.Attorney Docket No.1426.48.WO 29. The method of claim 28, wherein the prophylactic treatment is a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation.

30. The method of any one of the previous claims, wherein the subject is identified as having an increased risk of cancer aggressiveness, and the method further comprises administering a treatment course based on the increased risk of cancer aggressiveness.

31. The method of claim 30, wherein the treatment course is a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation based on the increased risk of cancer aggressiveness.

32. The method of any one of the previous claims, wherein the subject has cancer, and the method further comprises the step of monitoring the subject at a higher frequency for detection of cancer recurrence, cancer aggressiveness, and / or metastases relative to a subject without increased risk of cancer aggressiveness and / or recurrence.

33. The method of claim 32, wherein the step of monitoring comprises imaging the subject.

34. A method of monitoring cancer progression in a subject, the method comprising detecting histone gene expression at one or more replication-coupled histone genes on samples obtained at two more points in time from the same subject; and comparing the level of histone gene expression in each sample to a reference and / or to each other, wherein an increase in the level of histone gene expression in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression in the subject.Attorney Docket No.1426.48.WO 35. The method of claim 34, further comprising administering a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation to the subject with cancer progression.

36. The method of claim 34, wherein detecting histone gene levels comprises subjecting the sample to a nucleic acid amplification (e.g., qRT-PCR).

37. The method of claim 34, wherein detecting histone gene expression comprises detecting RNAPII levels at the one or more replication-coupled histone genes on samples obtained at two or more points in time from the same subject; and comparing an amount of the levels of RNAPII at the one or more replication-coupled histone genes in each sample to the reference and / or to each other, wherein an increase in the levels of RNAPII in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression.

38. The method of claim 37, wherein detecting RNAPII levels comprises subjecting the sample to CUTAC with an affinity agent comprising an antibody to a phosphoform of the C-terminal domain of RNA polymerase II (RNAPII), such as RNAPII-Ser2, RNAPII- Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.

39. A method of monitoring cancer progression in a subject, the method comprising detecting chromatin accessibility at one or more replication-coupled histone genes on samples obtained at two more points in time from the same subject; and comparing the level of chromatin accessibility in each sample to a reference and / or to each other, wherein an increase in the level of chromatin accessibility in the sample taken at a second point in time compared to the reference or to the sample at a first point of time is indicative of cancer progression in the subject.Attorney Docket No.1426.48.WO 40. The method of claim 39, further comprising administering a cancer biologic, RNA therapeutic, chemotherapy, surgery, and / or radiation to the subject with cancer progression.

41. The method of claim 39, wherein detecting chromatin accessibility comprises performing CUTAC.

42. The method of any one of claims 34-41, wherein the one or more histone genes comprises, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 of HIST2H3D, HIST2H4B, HIST2H3A, HIST2H2AA4, HIST2H2BC, HIST2H2AA4, HIST2H3A, HIST2H4B, HIST2H2AC, HIST2H2AB, HIST1H2AA, HIST1H2BA, HIST1H2APS1, HIST1H1A, HIST1H3A, HIST1H4A, HIST1H4B, HIST1H3B, HIST1H2AB, HIST1H2BB, HIST1H3C, HIST1H1C, HIST1H4C, HIST1H1T, HIST1H2BC, HIST1H2AC, HIST1H1E, HIST1H2BD, HIST1H2BE, HIST1H4D, HIST1H2AD, HIST1H2BF, HIST1H4E, HIST1H2BG, HIST1H2AE, HIST1H3E, HIST1H1D, HIST1H4F, HIST1H4G, HIST1H3F, HIST1H2BH, HIST1H3G, HIST1H2BI, HIST1H4H, HIST1H2BJ, HIST1H2AG, HIST1H4I, HIST1H2AH, HIST1H2BL, HIST1H2AI, HIST1H3H, HIST1H2AJ, HIST1H2BM, HIST1H4J, HIST1H4K, HIST1H2AK, HIST1H2BN, HIST1H2AL, HIST1H1B, HIST1H3I, HIST1H4L, HIST1H3J, HIST1H2AM, HIST1H2BO, or any combination thereof.

43. The method of claim 42, wherein the one or more histone genes comprises one or more of gene Reference Sequences: NM_001123375, NM_001034077, NM_001005464, NM_001040874, NR_036461, NM_001040874 _ chr1_149822627, NM_001005464_ chr1_149824180, NM_001034077_ chr1_149832329, NM_003517, NM_175065, NM_170745, NM_170610, NR_045125, NM_005325, NM_003529, NM_003538, NM_003544, NM_003537, NM_003513, NM_021062, NM_003531, NM_005319, NM_003542, NM_005323, NM_003526, NM_003512, NM_005321, NM_021063,Attorney Docket No.1426.48.WO NM_003523, NM_003539, NM_021065, NM_003522, NM_003545, NM_003518, NM_021052, NM_003532, NM_005320, NM_003540, NM_003547, NM_021018, NM_003524, NM_003534, NM_003525, NM_003543, NM_021058, NM_021064, NM_003495, NM_080596, NM_003519, NM_003509, NM_003536, NM_021066, NM_003521, NM_021968, NM_003541, NM_003510, NM_003520, NM_003511, NM_005322, NM_003533, NM_003546, NM_003535, NM_003514, NM_003527, or any combination thereof.

44. The method of any one of claims 34-43, wherein the sample comprises tissue, blood, or cell-free DNA.

45. The method of any one of claims 34-44, wherein the sample is from a tumor.

46. The method of any one of claims 34-45, wherein the sample is a formalin-fixed paraffin- embedded (FFPE) sample.

47. The method of any one of claims 34-46, wherein the reference is from non-cancerous tissue.

48. The method of any one of claims 34-47, wherein the reference is a threshold value.

49. The method of any one of claims 34-48, wherein the reference is based on a population of subjects.

50. The method of any one of claims 34-49, further comprising contacting the sample with a known amount of spike-in DNA configured to facilitate calibration.Attorney Docket No.1426.48.WO 51. The method of any one of claims 34-50, wherein the samples is from a subject with brain, breast, colon, liver, lung, stomach, kidney or rectum cancer.

52. The method of claim 51, wherein the brain cancer is meningioma.

53. The method of any one of claims 34-52, further comprising performing a chromatin assay on a biological sample.

54. The method of any one of claims 34-53, further comprising performing a chromatin accessibility assay on a biological sample.

55. The method of claim 54, wherein the chromatin assay is selected from a Cleavage Under Targeted Accessible Chromatin (CUTAC) assay, an Assay for Transposase-Accessible Chromatin (ATAC)-seq assay, a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-seq assay, a Nicking Enzyme-Assisted Accessible Chromatin Sequencing (NicE)-Seq assay or a Reverse Transcribe and Tagment (RT&Tag) assay.

56. The method of claim 55, comprising performing CUTAC wherein an affinity agent comprises an antibody to a phosphoform of the C-terminal domain of RNA polymerase II (RNAPII), such as RNAPII-Ser2, RNAPII-Ser5, RNAPII-Ser7, RNAPII-Ser2 / 5, or RNAPII-Ser5 / 7.

57. The method of any one of claims 34-56, further comprising detecting RNAPII at one or more enhancers.Attorney Docket No.1426.48.WO 58. The method of any one of claims 34-57, wherein the method is performed on solid support.

59. The method of claim 58, wherein the solid support is a bead, a slide, or a well (e.g., a microwell or nanowell).

60. The method of any one of claims 34-59, wherein progression comprises cancer recurrence or metastases.

61. The method of any one of claims 34-60, wherein the RNA therapeutic is specific for one or more target mRNAs.

62. The method of claim 61, wherein the one or more target mRNAs is an mRNA transcribed from one or more of the replication-coupled histone genes or one or more mRNAs translated into protein components that function within the histone locus body or target the U7 small nuclear RNA, or any combination thereof.

63. The method of claim 62, wherein the one or more replication-coupled histone genes encodes histone H1.

64. The method of Claim 62, wherein the one or more replication-coupled histone genes encodes histone H2A.

65. The method of claim 62, wherein the one or more replication-coupled histone genes encodes histone H2B.Attorney Docket No.1426.48.WO 66. The method of claim 62, wherein one or more replication-coupled histone genes encodes histone H3.

67. The method of claim 62, wherein the one or more replication-coupled histone genes encodes histone H4.

68. The method of claim 62, wherein the component that functions within the histone locus body is nuclear protein at the ataxia-telangiectasia locus (NPAT).

69. The method of claim 62, wherein the component that functions within the histone locus body is stem-loop binding protein (SLBP).

70. The method of claim 62, wherein the component that functions within the histone locus body is U7 small nuclear ribonuclear protein.

71. The method of claim 62, wherein the component that functions within the histone locus body is the human homolog of Drosophila melanogaster FLICE-associated huge protein (FLASH).

72. The method of any one of claims 62-71, wherein the component that functions within the histone locus body is a component of the histone cleavage complex (HCC).

73. The method of any one of claims 62-72, wherein the one or more therapeutic RNAs is a small interfering RNA (siRNA).Attorney Docket No.1426.48.WO 74. The method of any one of claims 62-72, wherein the one or more therapeutic RNAs is a hairpin RNA.

75. The method of any one of Claims 62-72, wherein the one or more therapeutic RNAs is an anti-sense RNA.

76. The method of any one of Claims 62-72, wherein the one or more therapeutic RNAs is a decoy oligonucleotide.

77. A method of treating cancer in a subject comprising introducing one or more therapeutic RNAs specific for one or more target mRNAs to a subject in need thereof, wherein the one or more target mRNAs is an mRNA transcribed from one or more replication- coupled histone genes or one or more mRNAs translated into protein components that function within the histone locus body or target the U7 small nuclear RNA, or any combination thereof.

78. The method of claim 77, wherein the one or more replication-coupled histone genes encodes histone H1, H2A, H2B, H3 and / or H4.

79. The method of claim 77, wherein the component that functions within the histone locus body is nuclear protein at the ataxia-telangiectasia locus (NPAT), stem-loop binding protein (SLBP), U7 small nuclear ribonuclear protein, the human homolog of Drosophila melanogaster FLICE-associated huge protein (FLASH), and / or a component of the histone cleavage complex (HCC).

80. The method of any one of claims 77-79, wherein the one or more therapeutic RNAs is a small interfering RNA (siRNA).Attorney Docket No.1426.48.WO 81. The method of any one of claims 77-79, wherein the one or more therapeutic RNAs is a hairpin RNA.

82. The method of any one of claims 77-79, wherein the one or more therapeutic RNAs is an anti-sense RNA.

83. The method of any one of claims 77-79, wherein the one or more therapeutic RNAs is a decoy oligonucleotide.

84. A kit comprising primers and / or probes specific to one or more replication-coupled histone genes and a reverse transcriptase.

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