Clonal Fitness Research Articles

Single-cell decoding of drug induced transcriptomic reprogramming in triple negative breast cancers

BackgroundThe encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states.ResultsWe present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure.ConclusionsWithin a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration, and re-treatment strategies.

PKD1 mutant clones within cirrhotic livers inhibit steatohepatitis without promoting cancer

Somatic mutations in non-malignant tissues are selected for because they confer increased clonal fitness. However, it is uncertain whether these clones can benefit organ health. Here, ultra-deep targeted sequencing of 150 liver samples from 30 chronic liver disease patients revealed recurrent somatic mutations. PKD1 mutations were observed in 30% of patients, whereas they were only detected in 1.3% of hepatocellular carcinomas (HCCs). To interrogate tumor suppressor functionality, we perturbed PKD1 in two HCC cell lines and six invivo models, in some cases showing that PKD1 loss protected against HCC, but in most cases showing no impact. However, Pkd1 haploinsufficiency accelerated regeneration after partial hepatectomy. We tested Pkd1 in fatty liver disease, showing that Pkd1 loss was protective against steatosis and glucose intolerance. Mechanistically, Pkd1 loss selectively increased mTOR signaling without SREBP-1c activation. In summary, PKD1 mutations exert adaptive functionality on the organ level without increasing transformation risk.

Abstract 1494: Clonal dominance defines metastatic dissemination in pancreatic cancer

Abstract Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically-implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and non-metastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients’ survival. Citation Format: I-Lin Ho, Chieh-Yuan Li, Fuchenchu Wang, Li Zhao, Jingjing Liu, Er-Yen Yen, Charles A. Dyke, Rutvi Shah, Zhaoliang Liu, Ali Osman Çetin, Francesca Citron, Sergio Attanasio, Virgina Giuliani, Tim Heffernan, Kim-Anh Do, Gaetano Gargiulo, Giulio Draetta, Alessandro Carugo, Ruitao Lin, Andrea Viale. Clonal dominance defines metastatic dissemination in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1494.

Clonal dominance defines metastatic dissemination in pancreatic cancer.

Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.

Early Evolution in Cancer: A Mathematical Support for Pathological and Genomic Evidence in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (CCRCC) is an aggressive form of cancer and a paradigmatic example of intratumor heterogeneity (ITH). The hawk-dove game is a mathematical tool designed to analyze competition in biological systems. Using this game, the study reported here analyzes the early phase of CCRCC development, comparing clonal fitness in homogeneous (linear evolutionary) and highly heterogeneous (branching evolutionary) models. Fitness in the analysis is a measure of tumor aggressiveness. The results show that the fittest clone in a heterogeneous environment is fitter than the clone in a homogeneous context in the early phases of tumor evolution. Early and late periods of tumor evolution in CCRCC are also compared. The study shows the convergence of mathematical, histological, and genomics studies with respect to clonal aggressiveness in different periods of the natural history of CCRCC. Such convergence highlights the importance of multidisciplinary approaches for obtaining a better understanding of the intricacies of cancer.

Abstract B080: Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer

Abstract The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into the clinic marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib – ORR = 37.1%, adagrasib - 43%, JDQ443 – 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFR, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a high-complexity DNA-barcoding system and mathematical modeling, we observed that pre-existing resistant clones to KRAS G12Ci gain clonal fitness during treatment independent of growth rate. Using single-cell profiling and RNA-lineage tracing barcoded systems, we identified distinct subpopulations of cells defined by specific chromatin and transcriptional states, including activation of interferon-gamma genes (ISG) and increased chromatin accessibility for pioneer transcription factors, which may contribute to MAPK reactivation-driven resistance. These results suggest a potential role of pioneer transcription factor-activated interferon-gamma response in driving MAPK feedback reactivation in KRAS G12Ci- resistant tumor subpopulations. Citation Format: Chendi Li, Christopher J Graser, Qian Qin, Usman M Syed, Barbara Karakyriakou, Sarah E Clark, Anne Y Saiki, Paul E Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron N Hata. Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B080.

Prevalence, Dynamics and Clinical Significance of Clonal Hematopoiesis of Indeterminate Potential (CHIP) in Newly Diagnosed Cancer Patients

INTRODUCTION Prevalence of CHIP in cancer patients (pts) is estimated at about 25%, its presence being associated with inferior outcomes and with increased risk of development of therapy-related myeloid neoplasms (TRMN). Despite the increased body of knowledge on cancer and CHIP, processes driving the selection of clones and their latter malignant transformation have not been fully elucidated. We hypothesized that CHIP in cancer pts might not only lead to TRMN but also affect the prognosis of the primary neoplasm and its treatment-related toxicity. Our study aims to describe the prevalence and dynamics of CHIP in treatment-naïve pts with cancer and to analyze its impact on clinical outcomes. METHODS This study included 103 pts with a first cancer diagnosis at age ≥ 60 years and eligible for anticancer treatment without prior exposure to cytostatic agents. Peripheral blood (PB) samples were collected at diagnosis and 6 months after treatment. A customized NGS panel covering common CHIP genes ( DNMT3A, TET2, ASXL1, JAK2, PPM1D, TP53, SF3B1, GNB1, SRSF2, CHEK2, CBL, GNAS, and NRAS) was used to identify CHIP-positive (VAF≥ 1%) and CHIP-negative cases. Clonal dynamics were assessed through NGS at 6 months after treatment, categorized as ‘growing’ when their VAF increased by more than 25% compared to the baseline, ‘shrinking’ when VAF decreased by more than 25% compared to the baseline and ‘stable’ if it remains unaltered. RESULTS Baseline characteristics are shown in table 1.The prevalence of CHIP in our cohort was 35%, with an average of 1.5 somatic variants per patient and 54 identified variants, and a median VAF of 4.7% (IQR 2.5-11.0%). Notably, mutations in DNMT3A (33%), TET2 (28%), and PPM1D (13%) were the most prevailing gene aberrations, accounting for nearly 75% of all variants, while other common CHIP genes such as ASXL1 (9%) or JAK2 (0%) were less frequent. TP53 variants represented 9% of all mutations, whereas SF3B1, GNB1, SRSF2, and CBL each accounted for 2%. Breast cancer pts displayed a significantly higher prevalence of CHIP compared to other primary neoplasms (66% vs. 36%, p=0.01) whereas no patient with bladder neoplasm presented CHIP at diagnosis (p=0.04). These observations were not warranted only by differences in the age or smoking habit of these subgroup of neoplasms. The mutational spectrum of CHIP across different cancer categories was comparable. Among 20 paired samples sequenced (baseline and post-genotoxic exposure), 41% of all variants exhibited a growing pattern, 31% a shrinking pattern, and 28% remained static (Figure 1). Platinum-based therapy exposition promoted clonal expansion in DNMT3A mutations (p=0.03), while this effect was not observed in PPM1D or other genes, likely due to the low sample size. Age, tobacco use, and type of primary neoplasm did not appear to influence clonal fitness. There were no significant differences in the incidence of infectious complications or chemotherapy-induced hematologic toxicity between CHIP-positive and CHIP-negative cohorts. With the current follow-up time (16.8 months), the overall response (ORR) and complete response rates (CR) appeared comparable (ORR: 94% in CHIP-positive vs. 85% in CHIP-negative; CR: 79% vs. 70%, respectively). A patient with diffuse large B-cell lymphoma and CHIP ( ASXL1, PPM1D, and TP53 variants) developed a TRMN (MDS-MD) 7 months after completing R-CHOP treatment. CONCLUSION The prevalence of CHIP in our cancer pts cohort is 35%, with breast cancer cases displaying a CHIP occurrence around 62% not previously reported. Our study highlights an enrichment of mutations in PPM1D in treatment-naïve cancer pts, surpassing the frequency of ASXL1 in contrast to prior literature. Genotoxic therapy promotes clonal expansion in 41% of variants in our cohort; although factors influencing CHIP fitness remain poorly understood, DNMT3A showed heightened susceptibility to platinum therapy. Finally, and in contrast contrast with our initial hypothesis, we found no evidence of impaired outcomes in the CHIP population. These results emphasize the need for further longitudinal follow-up. Acknowledgements: This work was supported by two grants from the Instituto de Salud Carlos III (PI20/00881 and PI 20/00531)(Co-funded by European Regional Development Fund. ERDF, a way to build Europe). 2021 SGR 00560 (GRC) Generalitat de Catalunya; economical support from CERCA Programme.

Repurposing GSK3B Small Molecule Inhibitors for Refractory Lymphoid Malignancies

Background: Small molecule GSK3B inhibitors were developed for solid tumors and neurological conditions, including Alzheimer's and Parkinson disease. Five GSK3B inhibitors achieved favorable safety and PK/PD profiles in a total of 22 Phase 1 and 2 clinical trials but no clinical response for any of these indications. GSK3B is the kinase that phosphorylates β-catenin to initiate its degradation, hence GSK3B small molecule inhibition results in rapid nuclear accumulation of β-catenin protein. Significance: Activating mutations in the Wnt/β-catenin pathway are common oncogenic drivers throughout all main types of cancer (n=64,812) but not found in B- and T-lymphoid malignancies (n=2,137). Studying tissue microarrays by β-catenin immunohistochemistry, lung, colon, breast, and melanoma specimen (n=57) showed consistently high levels of nuclear β-catenin, whereas B- and T-cell lymphomas (n=84) lacked β-catenin signal. Despite comparable mRNA levels, proteomic analyses revealed 60-400-fold lower β-catenin levels in lymphoid compared to epithelial tumors (n=1,389) suggesting that lymphoid malignancies rely on powerful mechanisms for β-catenin degradation, e.g. by GSK3B. Results: To study the effects of β-catenin activation in human cells, we induced expression of stabilized form of β-catenin in 18 lymphoid (B- and T-ALL, MCL, DLBCL, PTCL) and four myeloid leukemia cell lines. In B- and T-lymphoid cells, β-catenin accumulation compromised clonal fitness, colony formation and induced cell death, but increased competitive fitness and colony formation in myeloid leukemia cells. CEBPα-mediated reprogramming of B-ALL cells into a myeloid phenotype reversed the deleterious effects highlighting that effects of β-catenin-accumulation strictly depend on lymphoid lineage-identity. RNA-seq studies upon inducible activation of β-catenin in lymphoid cells, revealed transcriptional repression of Myc and Myc target genes as principal gene expression change. Unlike activating β-catenin:TCF7 complexes in epithelial cells, our global interactome studies in B- and T-ALL and lymphoma cells identified repressive β-catenin complexes with lymphoid-specific Ikaros zinc finger (IKZF1/2/3) proteins and several components of the NuRD complex including Mta1/2, Gatad2a/b, Chd4, HDAC1/2. Interactome and ChIP-seq analyses revealed that β-catenin together with lymphoid-specific Ikaros factors recruited repressive NuRD complex members to deacetylate H3K27 and repress MYC superenhancer (SE) regions. Genetic deletion of both IKZF1 and IKZF3 or HDRT-based mutation of Ikaros binding motifs within the Myc-SE enabled resistance to β-catenin accumulation. To leverage this previously unrecognized vulnerability of lymphoid malignancies, we examined five clinically approved GSK3B small molecule inhibitors that achieved favorable safety profiles at micromolar plasma concentrations in clinical trials for neurological disorders and solid tumors. Strikingly, LY2090314 and CHIR99021 were effective at low nanomolar concentrations in B- and T-cell malignancies that expressed lymphoid Ikaros factors. At IC50 concentrations of <5 nM, GSK3B-inhibitors induced massive accumulation of β-catenin, repression of MYC and acute cell death. In B-ALL cells with IKZF1-deletion, expression of the remaining IKZF3 factor was sufficient to retain full sensitivity to GSK3B-inhibition. Deletion of β-catenin, however, abolished the effects of GSK3B inhibitors and relieved suppression of MYC, demonstrating accumulation of β-catenin represents as central mechanism of action. Preclinical in vivo treatment experiments of refractory B-ALL and mantle cell lymphoma PDX validated small molecule GSK3B-inhibition as strategy to overcome conventional mechanisms of drug-resistance. Conclusions: B- and T-lymphoid malignancies not only lacked expression of β-catenin but critically depend on GSK3B for effective β-catenin degradation. Our interactome studies in lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros and NuRD complex factors. Strikingly, GSK3B small molecule inhibitors engage this new pathway at low nanomolar concentrations and demonstrated favorable safety profiles in Phase 1 and Phase 2 trials. Safety and efficacy of GSK3B-inhibitors opens up an immediate path for repurposing these agents towards refractory B-cell malignancies.

Clonal Hematopoiesis in Whole-Blood and Cell-Free DNA of Ovarian Cancer Patients Undergoing PARP-Inhibitor Treatment: An Exploratory Analysis of the ENGOT-ov48/Eudario Trial

Clonal hematopoiesis (CH), characterized by the expansion of somatically mutated hematopoietic stem cells, is an age-related phenomenon associated with the development of hematologic malignancies, increased cardiovascular risk, and other age-related proinflammatory conditions. In patients with solid tumors, cytotoxic therapies differentially select for clones with mutations in the DNA damage response (DDR) pathway, i.e. TP53 and PPM1D, leading to an elevated risk for therapy-related myeloid malignancies (t-MNs). Poly(ADP-ribose) polymerase inhibitors (PARPi) are frequently used to treat ovarian cancer with particularly high efficacy reported in tumors exhibiting homologous recombination (HR) deficiency caused by mutations in BRCA1/2 or other HR pathway genes. However, recent data indicate that PARPi treatment is also associated with an elevated risk for t-MNs. Here, we performed an in-depth analysis of CH in whole-blood (WB) and plasma of patients with relapsed high-grade ovarian cancer (HGOC) receiving platinum-based chemotherapy and PARPi treatment, elucidating the mutational landscape of CH, gene-specific dynamics, as well as the potential interference of CH with putative tumor-derived mutations in cell-free DNA (cfDNA). Serial specimen of 103 patients with relapsed, platinum-sensitive HGOC participating in the European Trial on Enhanced DNA Repair Inhibition in Ovarian Cancer (EUDARIO) study (ENGOT-ov48/NCT03783949) were analyzed using error-corrected targeted sequencing with a custom panel covering 45 myeloid and 27 HR-related genes. Study treatment consisted of six cycles of carboplatin-based chemotherapy followed by maintenance therapy with Niraparib ± the HSP90 inhibitor Ganetespib. A total of 128 somatic mutations with variant allele frequencies (VAFs) ≥ 1% were identified in 58 patients (56%) with a high fraction of DDR mutations (51 PPM1D mutations in 27 patients, 7 TP53 mutations in 7 patients). Age (median 62 years) and the number of previous therapy lines (median 1, range 1-5 lines) were significantly associated with the presence of CH. Specifically, prior exposure to PARPi treatment was strongly associated with both the presence of CH and the number of CH mutations. CH-positive patients had significantly shorter progression-free survival (median 7.9 months vs 10.6 months, p = 0.021 in log-rank test), and a trend for shorter overall survival (median 21.1 months vs 27.7 months, p = 0.16) in univariate survival analysis. With respect to adverse events, CH-positive patients more frequently had infections during study treatment (49% vs 26%, p = 0.025). All but 5 CH mutations with VAF ≥ 1% were also detectable in cfDNA with significant correlation of VAFs (R = 0.84, p < 0.0001). Mutations occurring solely in cfDNA (putatively tumor-derived) had a high fraction of TP53 and HR-related mutations, while DNMT3A and PPM1D, were rarely mutated (Figure 1). However, 23/87 (26%) of TP53 mutations and 31/98 (32%) of mutations in HR-related genes detected in cfDNA were of hematopoietic origin, underlining the importance of parallel sequencing of WB in liquid biopsies to avoid false positive results. The dynamics of CH clones under carboplatin and PARPi treatment were assessed in paired WB samples at initiation/end of treatment, which were available for 62 patients. 96 clones that initially had a VAF < 1% emerged during the treatment, including 56 PPM1D- and 10 TP53-mutated clones. Gene-wise comparison revealed a significantly higher median clonal fitness in PPM1D- and TP53-mutated clones compared to TET2- or DNMT3A-mutated clones (Figure 2). PPM1D-mutated clones exhibited lower fitness during PARPi treatment compared to carboplatin treatment (p = 0.0015 in Wilcoxon rank sum test), which was not the case for TP53-mutated clones. Finally, 8 samples with multiple mutations were subjected to single-cell genotyping on the Mission Bio Tapestri platform to investigate their clonal architecture. Preliminary data indicate clonal exclusiveness of co-occurring DDR mutations. In summary, our data reveals a high prevalence of CH in patients with relapsed HGOC and provides novel insights into the clonal architecture and dynamics of CH under carboplatin and PARPi treatment with a differential selection of DDR-driven clones. Moreover, we report a relevant interference of CH-derived mutations with tumor-derived mutations in cfDNA.

STAT5-Feedback Controls Distinct Metabolic States for Dynamic Transitions between Cellular Activation and Quiescence in Acute Lymphoblastic Leukemia

Background and significance: B-cell acute lymphoblastic leukemia (B-ALL) is characterized by dynamic shifts between activation and quiescence. These cycles are predicated by fluctuations in oncogenic signaling, which mimic pre-BCR and cytokine receptor signaling, e.g. IL7 receptor signaling and phosphorylation of STAT5. These fluctuations in cytokine receptor and STAT5 signaling are mitigated by powerful feedback regulators, including CISH and SOCS (suppressors of cytokine signaling) family members. While B-ALL cells depend on oncogenic STAT5 signaling, CISH and SOCS2 negative feedback regulators of STAT5 are highly expressed in aggressive B-ALL and high expression levels are strongly linked to poor clinical outcome, positive MRD and increased likelihood of bone marrow relapse in children (COG P9906) and adults (ECOG E2993). To determine whether high levels of CISH and SOCS2 are biomarkers of high oncogenic STAT5 signaling or mechanistically contribute to clonal fitness and adaptability of B-ALL, we performed genetic experiments in mouse models for B-ALL. Here we examined the mechanisms and consequences of active and inactive states of STAT5 by studying constitutively active (CA) and non-phosphorylatable Y694F (YF) mutations of STAT5. Results: Consistent with a central role of STAT5-feedback control in B-ALL survival, inducible ablation of Cishfl/fl resulted in rapid depletion of B-ALL cells. Socs2-/- B-ALL cells rapidly underwent cellular senescence, lacked colony forming and leukemia-initiating capacity in serial transplantation experiments. Likewise direct interference with STAT5-function, by mutations resulting in constitutive activation (STAT5-CA) or an inactive state (STAT5-YF). Consistent with a requirement of adaptive STAT5-feedback control in B-ALL, both Stat5-CA and Stat5-YF resulted in rapid cell death. Induction of Stat5-CA caused accumulation of biomass indicated by their increased cell size, whereas Stat5-YF caused rapid cell shrinkage ( Figure A). To determine whether the mTOR cell growth pathway was responsible for cell death induced by Stat5-CA, we tested whether the genetic deletion of Mtor or pharmacological inhibition of protein synthesis were sufficient to rescue B-ALL cell death. As expected, B-ALL cells retaining intact Mtor rapidly underwent energy crisis and cell death, while genetic deletion of Mtor reversed the toxic effects of Stat5-CA, suggesting that Stat5-CA is associated with excessive protein synthesis and defective ER turnover. Indeed, Stat5-CA increased protein synthesis rate and ER content compared to EV control, and these effects were opposed by Stat5-YF. To compare the metabolic outcomes of Stat5-CA and Stat5-YF, we performed mass spectrometry-based metabolomic analyses ( Figure B). Consistent with the role of mTOR in promoting glycolysis, Stat5-CA increased the content of glycolytic intermediates in B-ALL cells. In contrast, Stat5-YF increased phospholipid intermediates and phosphatidylethanolamine (PtdEtn). Opposed by Myc, Stat5-YF increased the expression levels of Bcl6 and autophagic genes, suggesting that Bcl6-dependent programs promote PtdEtn-LC3B conjugation, thereby autophagosome formation. As expected, microscopic validation with LC3B-GFP reporter revealed that Stat5-YF increased the number of LC3B-GFP puncta, upon pharmacological inhibition of autophagosome-lysosome fusion ( Figure A). Consistent with STAT5-CA promoting Myc- as opposed to STAT5-YF driving Bcl6-dependent programs, a flow cytometry analysis of Myc-eGFP/Bcl6-mCherry dual reporter expression in murine B-ALL cells demonstrated that changes between glycolytic Myc+ and autophagic Bcl6+ states were the direct consequence of Stat5-CA and Stat5-YF induction, respectively. Conclusions: Our results demonstrate feedback regulators of STAT5, including SOCS2 and CISH are not only biomarkers of increased oncogenic STAT5 activity and a more aggressive course of disease linked to poor overall outcomes. Importantly, negative STAT5 feedback control mechanisms are essential to enable nimble adaptations of B-ALL cells in response to fluctuations of oncogenic signaling strength and availability of nutrients during distinct metabolic states of cellular activation (MYC) and quiescence (BCL6).

SETD2 mutations do not contribute to clonal fitness in response to chemotherapy in childhood B cell acute lymphoblastic leukemia

Mutations in genes encoding epigenetic regulators are commonly observed at relapse in B cell acute lymphoblastic leukemia (B-ALL). Loss-of-function mutations in SETD2, an H3K36 methyltransferase, have been observed in B-ALL and other cancers. Previous studies on mutated SETD2 in solid tumors and acute myelogenous leukemia support a role in promoting resistance to DNA damaging agents. We did not observe chemoresistance, an impaired DNA damage response, nor increased mutation frequency in response to thiopurines using CRISPR-mediated knockout in wild-type B-ALL cell lines. Likewise, restoration of SETD2 in cell lines with hemizygous mutations did not increase sensitivity. SETD2 mutations affected the chromatin landscape and transcriptional output that was unique to each cell line. Collectively our data does not support a role for SETD2 mutations in driving clonal evolution and relapse in B-ALL, which is consistent with the lack of enrichment of SETD2 mutations at relapse in most studies.

Clonal hematopoiesis and inflammation - the perpetual cycle.

Acquired genetic or cytogenetic alterations in a blood stem cell that confer clonal fitness promote its relative expansion leading to clonal hematopoiesis (CH). Despite a largely intact hematopoietic output, CH is associated with a heightened risk of progression to hematologic malignancies and with non-hematologic health manifestations, including cardiovascular disease and overall mortality. We focus on the evidence for the role of inflammation in establishing, maintaining and reciprocally being affected by CH. We describe the known pro-inflammatory signals associated with CH and preclinical studies that elucidated the cellular mechanisms involved. We review the evolving literature on early-onset CH in germline predisposition conditions and the possible role of immune dysregulation in this context.

Immune mechanisms shape the clonal landscape during early progression of prostate cancer.

Understanding the role of the immune microenvironment in modulating intratumor heterogeneity is essential for effective cancer therapies. Using multicolor lineage tracing in genetically engineered mouse models and single-cell transcriptomics, we show that slowly progressing tumors contain a multiclonal landscape of relatively homogeneous subpopulations within a well-organized tumor microenvironment. In more advanced and aggressive tumors, however, the multiclonal landscape develops into competing dominant and minor clones accompanied by a disordered microenvironment. We demonstrate that this dominant/minor landscape is associated with differential immunoediting, in which minor clones are marked by an increased expression of IFNγ-response genes and the Tcell-activating chemokines Cxcl9 and Cxcl11. Furthermore, immunomodulation of the IFNγ pathway can rescue minor clones from elimination. Notably, the immune-specific gene signature of minor clones exhibits a prognostic value for biochemical recurrence-free survival in human prostate cancer. These findings suggest new immunotherapy approaches for modulating clonal fitness and tumor progression in prostate cancer.

Abstract 6625: Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia

Abstract Acute lymphoblastic leukemia (cALL) is the most common pediatric cancer, and since up to 20% of children relapse after initially responding to chemotherapy treatment, the largest cause of cancer-related childhood deaths in the western world. With the idea that dissecting the evolutionary population dynamics leading to relapse would help explain treatment failure from a mechanistic standpoint, several studies have performed comparisons of genetic heterogeneity at diagnosis and relapse. These showed that as many as 70% of relapses are dominated by a single diagnostic subclone or one of its evolutionary progeny; leading to the prevailing view that treatment selection in cALL primarily operates at the genotype level. However, a crucial caveat of these studies is their reliance, due primarily to technical and economic constraints, on very limited data timepoints. While the dynamics linking said timepoints are often interpreted based on simple intuition. To overcome this sparse data challenge and reconstruct the entire spatial and temporal evolutionary trajectories of individual cALL cells from diagnosis to relapse, we have integrated experimental data with an in silico model. Single-cell WGS and RNAseq data were obtained from primary and xenografted leukemic cells before and after chemotherapy. These were then used to parameterize an agent-based mathematical model allowing tracking of the genotype and evolving phenotype of individual cells over time. When short treatment courses akin to those we delivered in vivo were used, the in silico model closely recapitulated the experimental results. Since cALL treatment lasts several years, it cannot be entirely modelled in vivo. Due to the rarity of post-treatment residual cells and the lack of universal cell surface markers to isolate them, direct analysis of clinical samples is also challenging. However, based on the initial treatment data, our calibrated model provided a unique opportunity to simulate longer remission-relapse dynamics of several leukemias. Mechanistic analysis of the collected data showed that, strikingly, in most cases, clonal dominance at relapse results from stochastic sweeps rather than deterministic differences in clonal fitness. Indeed, independent treatment of the same leukemia with an identical drug regimen more frequently ended with a considerably different clonal landscape (either polyclonal disease or a different dominant clone) than with the same outcome initially observed. Rather than genotype differences, the size of residual disease and a founder effect linked to the stochastic exit, post-treatment, of individual cells from quiescence were the key determinants of the relapse genetic landscape. Altogether, our data highlight the misinterpretation risk associated with snapshot analysis and might inform promising alternative treatment schedules. Citation Format: Virginia Turati, Javier Herrero, Mark Robertson-Tessi, Tariq Enver, Andriy Marusyk, Alexander RA Anderson. Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6625.

Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This invivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

Monitoring Plasma Cells and Clonal Emergence through Genome Sequencing of Circulating Tumor Cells for Minimally Invasive Molecular Characterization of Multiple Myeloma

Introduction Multiple Myeloma (MM) develops from well-defined precursor stages, however, bone marrow (BM) biopsy limits screening and monitoring strategies for patients. Precursor conditions are incidentally diagnosed and require invasive BM biopsies for complete characterization, highlighting the need for improved early detection methods. Minimally invasive blood biopsies to measure circulating tumor cells (CTCs) as markers of MM disease development are a promising solution to this need. CTCs are amenable to repeatable enrichment, enumeration, and molecular characterization for early detection, real-time monitoring, and assessment of treatment response and clonal evolution. We present our novel method, MinimuMM-seq, for enumeration and whole genome sequencing (WGS) of CTCs that enables genomic profiling and WGS-based cytogenetic analyses from enriched liquid biopsy samples. CTCs were enumerated from 241 patients, with 50 patients undergoing WGS that revealed that CTCs were of tumor origin and could faithfully match BM sequencing results, detecting 100% of clinically reported events. Longitudinal sampling of CTCs tracked clonal dynamics over time and detected the emergence of high-risk genetic subclones. Methods Peripheral blood from 241 precursor patients (82 MGUS, 159 SMM) from the PCROWD observational study (IRB #14-174) was collected in CellRescuepreservation tubes and processed on the CellSearch® system (Menarini Silicon Biosystems) using the Circulating Multiple Myeloma Cell (CMMC) assay kit with 4mL of blood. Enrichment and enumeration of CTCs was based on CD138+38+CD45-19- immunophenotype. Nucleated cells were identified using DAPI staining. In 50 patients, CTCs were sorted for library construction, quantification, and WGS on Illumina NovaSeq6000. Mutation analyses were performed with the cancer genome analysis pipelines of the Broad Institute. Tumor phylogeny was reconstructed with the PhylogicNDT algorithm in patients with matched CTC and BM sequencing (n=27), as well as serial CTCs derived from PB only (n=10). Results CTCs were detected in 75% of MGUS and 86% of SMM patients enrolled in the study, with a median count of 2.5 (range 0 to 1328) and 13 CTCs (range 0 to 43836), respectively. An increased number of CTCs correlated with progressive disease staging from MGUS, to low, intermediate and high-risk SMM defined by IMWG 2/20/20 risk stratification. MinimuMM-seq yielded a median tumor purity of 98% (range 45 to 100%) in CTCs. Our method detected 100% of clinically reported BM biopsy events by fluorescence in situ hybridization (FISH), including translocations and copy number abnormalities. It could replace molecular cytogenetics for diagnostic yield and risk classification in patients with paired BM and CTC samples (n=27). Additional diagnostic yield compared to clinical BM FISH was found in 88% of patients. Our method detected novel events and unknown/cryptic translocations, such as t(8;14) and t(14;20), and MM driver mutations, including KRAS, NRAS, and TP53. As peripheral blood continuously circulates, liquid biopsy sampling may be affected by the phenomena of spatial and temporal heterogeneity in CTC burden. In 10 patients longitudinal CTC sampling was carried out to monitor clonal evolution of disease. We validated stability of the major clone and described shifting dynamics of subclones over time with the emergence of potentially high-risk CTC subclones. Expansion of subclones harboring MM events (such as 13q deletion) and driver mutations (such as KRAS and DIS3) was observed, indicating their potential to confer selective advantage for clonal fitness and independence for circulation. Anecdotally, one SMM patient received early interventional treatment, and we identified the emergence of the new fittest clone (bearing KRAS p.G12S) before any potential resistance was clinically observed. Conclusion We have established an approach for WGS of CTCs that can replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. We tracked clonal dynamics over time and detected the emergence of high-risk CTC subclones. Our findings show that capture and genomic profiling of CTCs could be a robust surrogate for BM biopsy, allowing minimally invasive detection and monitoring of disease and unlocking the clinical potential of liquid biopsies for MM diagnostics.

ERK2 Substrate Binding Domains Play Distinct Roles in Megakaryocytic-Erythroid Lineage Progression and Mediates Clonal Fitness in Myeloproliferative Neoplasms

ERK2 Substrate Binding Domains Play Distinct Roles in Megakaryocytic-Erythroid Lineage Progression and Mediates Clonal Fitness in Myeloproliferative Neoplasms

MODL-25. ANALYSIS OF TUMOR CELL HETEROGENEITY IN A HUMAN NEURAL PROGENITOR-BASED MODEL OF GLIOBLASTOMA REVEALS NEURODEVELOPMENTAL PROGRAMS AND CYTOSKELETAL DYNAMICS

Abstract Glioblastoma (GBM) is the most common primary malignant neoplasm with poor survival despite treatment. Developing effective therapies remains challenging due to intratumoral heterogeneity, which drives therapeutic resistance and recurrence. To understand how genetic events alter the epigenome to enhance clonal fitness, we developed an isogenic human neural progenitor cell (NPC)-based model of the proneural (PRO) GBM subtype. We introduced TERT promoter (TERTp) C228T and gain-of-function TP53 R248Q mutations in H1 human embryonic stem cells. Wildtype (WT) cells, single TERTp, and double TERTp/TP53 mutants underwent differentiation to NPCs. Lentiviral transduction of double mutants with PDGFRA D842V resulted in triple mutant PRO NPCs. Bulk and single cell transcriptomics of our model system revealed hundreds of gene expression changes with increased mesoderm and human GBM mesenchymal (MES) subtype signatures in single TERTp and double TERTp/TP53 mutants, versus WT cells. TERTp mutation increased telomerase expression and activity, conferring proliferative advantage and immortalization in NPCs and astrocytes. Additionally, TERT expression was further increased in TERTp/TP53 mutants. Surprisingly, triple mutant PRO NPCs, versus WT, displayed < 100 differentially expressed genes, associated with neurodevelopmental and dynamic cytoskeletal processes. Evolution analyses using gene counts signature and splicing dynamics revealed a developmental trajectory model from WT to additive mutants to PRO NPCs. Only triple mutant PRO NPCs formed tumors after intracranial injection in athymic nude mice, with mean survival of 100 days. Tumors presented histopathological features of GBM, and single cell transcriptomic analyses revealed evolution from immune-interacting to both neural progenitor- and neuronal-like subpopulations, with similar cell cycling signatures. Transcription factor genes related to WNT signaling and lineage commitment as well as glial and neuronal cytoskeletal genes exhibited epigenetic selection in vivo, signatures also observed in PRO NPCs in vitro. Our model thus provides opportunity for dissection of epigenetic and functional mechanisms underlying serial mutations during PRO tumor evolution.

Abstract 2228: A multi-omic perspective of how selection shapes blood cancer risk phenotypes in aging populations

Abstract The genetic architecture of blood is highly complex with germline polymorphisms, somatic point mutations, and larger chromosomal alterations playing a role in shaping the fitness of the immune cells. Many advances have been made in understanding how the age associated acquisition of point mutations and somatic structural variants (SSVs) in blood, termed Age-Related Clonal Hematopoiesis (ARCH), predispose individuals to hematological cancer or cardiovascular disease. Yet, ARCH is commonly observed in healthy individuals and our ability to predict who is at risk of progressing to disease remains limited. A previous study which integrated deep learning and population genetics methods to evaluate the complex interplay of selection on point mutations in deeply sequenced blood samples highlighted the role that negative selection plays in prevention progression to hematological cancer. Here, we interrogate the mutational and selective pressures within blood to better understand how the full spectrum of somatic changes within an individual impact clonal fitness and disease outcomes. We evaluate how selection shapes the prevalence of large somatic structural variation in blood sampled from 15,910 individuals across over 20 different genetic ancestries, including the Thousand Genomes Study and the Canadian Partnership for Tomorrow’s Health study. Using dense genotyping arrays, we capture SSVs among 20 populations and find that ARCH attributed to somatic structural variation is three times as high as previously reported with up to 14% of individuals harbouring at least one large SSV in their blood. We estimate the rate at which SSVs accrue in blood cells and find that selection impacts the size and frequency of SSVs within individual blood populations. To determine the functional impact of clonal mutations on molecular phenotypes, we investigate the relationship between structural variation and the transcriptome. We show that gains, losses and copy number neutral variants impact gene expression distinctly, with stabilizing selection shaping the penetrance of copy number alterations in gene expression. Our work shows how different classes of selection shape clonal dynamics in blood thus enabling us to better understand why certain individuals are at a high risk of malignancy. Citation Format: Kimberly Skead, David Soave, Marie-Julie Fave, Vanessa Bruat, Quaid Morris, Philip Awadalla. A multi-omic perspective of how selection shapes blood cancer risk phenotypes in aging populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2228.

Functionalized Lineage Tracing Can Enable the Development of Homogenization-Based Therapeutic Strategies in Cancer.

The therapeutic landscape across many cancers has dramatically improved since the introduction of potent targeted agents and immunotherapy. Nonetheless, success of these approaches is too often challenged by the emergence of therapeutic resistance, fueled by intratumoral heterogeneity and the immense evolutionary capacity inherent to cancers. To date, therapeutic strategies have attempted to outpace the evolutionary tempo of cancer but frequently fail, resulting in lack of tumor response and/or relapse. This realization motivates the development of novel therapeutic approaches which constrain evolutionary capacity by reducing the degree of intratumoral heterogeneity prior to treatment. Systematic development of such approaches first requires the ability to comprehensively characterize heterogeneous populations over the course of a perturbation, such as cancer treatment. Within this context, recent advances in functionalized lineage tracing approaches now afford the opportunity to efficiently measure multimodal features of clones within a tumor at single cell resolution, enabling the linkage of these features to clonal fitness over the course of tumor progression and treatment. Collectively, these measurements provide insights into the dynamic and heterogeneous nature of tumors and can thus guide the design of homogenization strategies which aim to funnel heterogeneous cancer cells into known, targetable phenotypic states. We anticipate the development of homogenization therapeutic strategies to better allow for cancer eradication and improved clinical outcomes.