In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities

Abstract

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.

Significance: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Figure 1.

Multiplexed CRISPR editing of CLL-LOF lesions generates CLL disease models. A, Schema for the generation of transplant lines. BM donor lines containing homozygous del(13q) and B cell–restricted Cas9-GFP [i.e., del(13q)-Cd19Cas9-GFP] were obtained by intercrossing del(13q)-Cd19cre/cre and del(13q)-Cas9/Cas9 mice. LSKs isolated from 8- to 12-week-old del(13q)-Cd19Cas9 animals were transduced in vitro with a pool of lentivirus-expressing sgRNAs against the 6 or 5 LOF mutations of interest (Atm, Mga, Samhd1, Chd2, Birc3, Trp53 in “6-plex”; Trp53 absent in “5-plex”) or corresponding nontargeting pools of scrambled sgRNAs and transplanted into 8- to 12-week-old immune-competent (CD45.1) or immune-deficient (NSG) recipients (n = 30–35/group). B, Single-cell qPCR-based analysis of sgRNA expression in LSK from the 3 targeting and the 3 nontargeting cohorts, as assessed at 72 hours after transduction and in vitro culture. The number of sgRNAs quantified in individual single cells is displayed. C, Percent (%) animals with circulating CLL in targeting and nontargeting cohorts. P, Fisher exact test. D, Longitudinal tumor burden assessments by flow-cytometric analysis of peripheral bleeds. A logistic mixed-effects model was used to estimate the average trajectories of peripheral longitudinal tumor burden (%GFP⁺mCh⁺B220⁺CD5⁺Igκ⁺ cells) in individual mice from the targeting (6-plex: dark brown; 5-plex: salmon) and nontargeting cohorts (gray). Disease onset and survival (median and range) for each cohort are indicated.

Figure 2.

Transformation into RS shows phenotypes consistent with human disease. A, Representative flow-cytometric plots highlighting the presence of small (CLL), small and large (CLL/RS), or large (RS) cells in the SP of one representative animal per disease pattern, at euthanasia. B, Representative H&E and CD5/PAX5 images from SP and BM of one case per group. Arrows indicate the presence of both small (blue) and large (red) cells in the representative CLL/RS case. Images captured at 10× magnification; black bar,100 μm; white bar, 20 μm. C, Pie charts displaying number of total cases of CLL (blue), CLL/RS (purple), and RS (red) in CD45.1, NSG 6-plex, NSG 5-plex and all nontargeting cohorts. Overall penetrance for each cohort is indicated. P, Fisher exact test. D, Disease burden as represented by %GFP⁺mCherry⁺B220⁺CD5⁺ Igκ⁺ cells in PB, SP, and BM of 11 NT-CLL, 21 CLL, 18 CLL/RS, and 25 RS cases at euthanasia. NT-CLL, nontargeting CLL controls. Horizontal lines, group median values. P values, ANOVA with Tukey correction for multiple comparisons. E, Number of cases displaying visible nodal infiltrations. LN, Lymph nodes. F, Representative IHC staining from one murine (left) and one human RS case (right) stained for CD5/PAX5, MUM1, BCL6, CD30, MYC, and Ki67. Images were taken at a 40× magnification; scale bar, 50 μm. G, Number of animals per pattern, and IGHV homology to germline, as assessed by BCR Immuno-Seq. H, Percent (%) clonotype frequency of shared BCR rearrangements as analyzed at time of CLL and upon RS transformation (euthanasia) in 2 CLL/RS and 3 RS cases sampled serially. The dominant RS clonotype is shown in purple (CLL/RS) and red (RS).

Figure 3.

Coselection of combinatorial traits is associated with RS transformation. A, Heat map showing CRISPR-edited lesions (in preleukemic normal B cells and at euthanasia) in relationship with disease pattern, recipient strain, and the number of introduced LOFs. Cases for which longitudinal CRISPR-seq, single-cell DNA-seq, WGS, and BCR-IGHV analyses were performed are indicated in green. Percent cases carrying clonal mutations (>85% indels) are shown in the bar diagram (blue, CLL; purple, CLL/RS; red, RS). B, Total number of clonal drivers in all cases with CLL (n = 21), CLL/RS (n = 18), and RS (n = 25). P value, ANOVA with Tukey correction for multiple comparisons. C, The proportion in which a recurrent driver is found as clonal or subclonal across the CLL, CLL/RS, and RS samples is provided (top), along with the individual % indels values (and group medians, horizontal lines) for each sample. P, Fisher exact test comparing the proportions of clonal and subclonal events in CLL versus CLL/RS or CLL versus RS, with Bonferroni correction for multiple comparisons. D, Number of mice carrying individual or selected combinations of clonal drivers, as analyzed by bulk CRISPR-seq at euthanasia. E, Longitudinal clonal trajectories of gene edits as analyzed by CRISPR-seq in 4 RS cases. Pre-leuk, preleukemic B cells. Clonality thresholds of 85% presence of indels are indicated by the horizontal dotted black line. Filled lines, clonal drivers; dotted lines, subclonal. F, Modification rates across single cells for the six on-target loci (columns) using single-cell amplicon sequencing. Splenocytes from 2 CLL/RS (n = 2,499 and 829) and 2 RS cases (n = 4,018 and 505) were sampled at euthanasia. A scale bar ranging from 0% (blue color) to 100% (red color) modifications is shown. G, Total number of chromosomal amplifications and deletions (right bars) in all cases with CLL (n = 5), CLL/RS (n = 4), and RS (n = 8) analyzed by WGS. P value, ANOVA with Tukey correction for multiple comparisons. H, Overlay of copy-number profiles from 5 del(13q)-CLLs, 2 CLL/RS 5-plex, 2 CLL/RS 6-plex, and 8 RS samples, as analyzed by WGS. The y axis shows the mean copy-number variant (CNV) ratio within a running window of 1-Mb pairs in each chromosome within the different cohorts. Recurrent human and murine RS drivers are indicated in the figure.

Figure 4.

Murine transcriptomes show strong similarity with human disease. A, Heat map of differentially expressed genes among the four different groups of mice (4 normal B, 5 del(13q)-CLL, 4 CLL/RS, and 11 RS). ANOVA FDR < 0.1 was used as a cutoff. B, Signaling pathways enriched for the differentially expressed genes in A. C, Concordantly modulated genes in human and murine RS and respective functional categories. P, Pearson correlation. D, Gene set enrichment plots showing the correlation of human RS versus CLL gene sets (upregulated, top; downregulated, bottom plot) in murine RS versus CLL data. NES, normalized enrichment score; FDR, false discovery rate. E, Representative IGV tracks of MYC ChIP-seq and RNA-seq data of two MYCMGA top targets in Mga-mutant RS and del(13q)-CLL. F, Representative IGV tracks of CHD2 ChIP-seq and RNA-seq data of two CHD2 top targets involved in the interferon γ pathway in Chd2-mutant RS and del(13q)-CLL. G, Log₂FC [RS vs. del(13q)-CLL] of MGA targets included in MYC targets and IFNγ response categories. The top 500 MGA cistromeGO targets were used in the analysis. H, Log₂FC [RS vs. del(13q)-CLL] of CHD2 targets included in E2F targets and IFNγ response categories. The top 500 CHD2 CistromeGO targets were used in the analysis. I, Immunoblot analysis of phospho-STAT1 and STAT3 (and respective totals), after 5- and 15-minute stimulation with soluble IFNα and IFNγ. One representative normal B-cell, del(13q)-CLL and RS sample is shown. GAPDH was probed, as control. J, Relative abundance of P-STAT1 and P-STAT3 levels across the three replicates of normal B cells, CLL and RS samples. Mean with SEM is displayed. P, ANOVA. K, Bar plot showing HLA class I and II MFI ratios (compared with isotype controls), as assessed by flow cytometry on normal B, del(13q)-CLL and RS cases (n = 5/group). Mean with SEM is displayed. P, ANOVA. L, Mean expression levels (in transcripts per million) of class I (HLA-A, -B, -C) and class II family (HLA-DR, -DQ) on RNA-seq data from 5 human CLL and matched RS cases. P, paired t test.

Figure 5.

Tonic PI3K signaling is a characteristic feature of RS. A, Immunoblot analysis of 12 murine RS and 14 human RS cases, assessed for baseline expression of c-MYC, PTEN, and phosphorylation and total expression of AKT and S6 kinases. GAPDH is shown as a loading control. Separate gels, run concomitantly, were used for the analysis. B, Heat map of relative phosphorylation intensities in AKT and S6, as analyzed by Western blot in del(13q)-CLL (n = 5), CLL/RS (n = 6), RS (n = 12), and human RS (n = 14). P, ANOVA. C, Linear regression of c-MYC and PTEN protein expression levels (normalized to GAPDH) in 12 murine and 14 human RS cases. P, Pearson correlation. D, Representative IGV tracks of Chd2 ChIP-seq and RNA-seq data of the Ptpn6 gene region from one representative normal B, one del(13q)-CLL, one Chd2-wt RS, and one clonal Chd2-mut RS case. E, Western blot analysis of SHP1 levels in primary murine RS carrying either wild-type (WT) or clonal Chd2 mutation (MUT). GAPDH is displayed, as control. F, Relative SHP1 expression (normalized to GAPDH) in 5 murine RS carrying subclonal/wt Chd2, and 5 cases carrying clonal Chd2 lesions. Mean with SEM is displayed. P, Mann–Whitney. G, Annotation of Uniform Manifold Approximation and Projection (UMAP) representation of single-cell chromatin accessibility profiles by cell type of murine data (left). B cells (black), CLL (blue), and RS (red) cells, T cells (brown), myeloid (gray), and erythroid (beige) compartment and deviation scores of TCF3 transcription factor motifs in murine data sets (right). H, Distribution of deviation z-scores of murine TCF3 binding motif across T cells, B cells, CLL, and RS. Median and interquartile ranges are displayed. P, ANOVA with Tukey correction for multiple comparisons. I, Annotation of UMAP representation of human single-cell chromatin accessibility profiles by cell type (left). B cells (black), CLL (blue), RS (lymph node, red; BM and PB, dark pink) cells, T cells (brown), and deviation scores of TCF3 transcription factor motifs (right). J, Distribution of deviation z-scores of human TCF3 binding motif across T cells, B cells, CLL, and RS. Median and interquartile ranges are displayed. P, paired t test. K, PI3K signature gene expression in the 4 normal B, 5 del(13q)-CLL, 4 CLL/RS, and 11 RS murine cases (mean with SEM) and in the 5 paired CLL/RS samples, analyzed by RNA-seq. P (murine), ANOVA; P (human), paired t test. L, Correlation between PI3K scores and MYC scores in the 4 CLL/RS and 11 RS murine cases and 37 human RS analyzed by RNA-seq. P value, Pearson correlation.

Figure 6.

MYC/mTOR/PI3K signaling are targetable vulnerabilities of RS. A, Summary heat map of drug treatment studies of murine primary splenocytes [7 del(13q)-CLL, 3 CLL/RS, and 12 RS] and 5 human RS cases. Percent (%) viability compared with the DMSO control is displayed. B, Bar plots showing percent (%) viability after JQ1, everolimus, duvelisib, palbociclib, and alisertib treatment. Mean with SEM is displayed. P, ANOVA. C, Correlation between response to duvelisib (% viability) and phospho-AKT levels, as detected by immunoblot in 5 del(13q)-CLL, 2 CLL/RS, 8 murine RS, and 14 human RS cases. P, Pearson correlation. D, Correlation between response to everolimus (% viability) and phospho-S6 levels on the same set of samples per C, as detected by immunoblot. P, Pearson correlation. E, Immunoblot assessment of c-MYC expression levels following a 12-hour treatment with single-agent duvelisib, everolimus, JQ1 or respective duvelisib-based combinations. One representative mouse and human samples are shown and GAPDH is used as loading control. F, Percent (%) viability normalized to DMSO control of 4 murine and 3 human RS primary specimens treated for 40 hours with single-agent duvelisib, everolimus, JQ1, or respective duvelisib-based combinations. Mean with SEM is displayed. P value, ANOVA with Tukey correction for multiple comparisons. G, Transplant schema for combination treatment studies and survival curve of NSG mice transplanted with RS splenocytes and then treated with 50 mg/kg duvelisib, 10 mg/kg everolimus, or combination (left), or 50 mg/kg duvelisib, 50 mg/kg JQ1, or combination (right) for 2 weeks followed by observation until survival endpoints. P values, log-rank test with Bonferroni correction for multiple comparisons. The pink shaded area indicates the treatment period.