High Proliferation Research Articles

3D printed polylactic acid-based nanocomposite scaffold stuffed with microporous simvastatin-loaded polyelectrolyte for craniofacial reconstruction

Critical sized craniofacial defects are among the most challenging bone defects to repair, due to the anatomical complexity and aesthetic importance. In this study, a polylactic acid/hardystonite-graphene oxide (PLA/HTGO) scaffold was fabricated through 3D printing. In order to upgrade the 3D printed scaffold to a highly porous scaffold, its channels were filled with pectin-quaternized chitosan (Pec-QCs) polyelectrolyte solution containing 0 or 20 mg/mL of simvastatin (Sim) and then freeze-dried. These scaffolds were named FD and FD-Sim, respectively. Also, similar PLA/HTGO scaffolds were prepared and dip coated with Pec-QCs solution containing 0 or 20 mg/mL of Sim and were named DC and DC-Sim, respectively. The formation of macro/microporous structure was confirmed by morphological investigations. The release of Sim from DC-Sim and FD-Sim scaffolds after 28 days was measured as 77.40 ± 5.25 and 86.02 ± 3.63 %, respectively. Cytocompatibility assessments showed that MG-63 cells had the highest proliferation, attachment and spread on the Sim containing scaffolds, especially FD-Sim. In vivo studies on a rat calvarial defect model revealed that an almost complete recovery occurred in the group treated with FD-Sim scaffold after 8 weeks and the defect was filled with newly formed bone. The results of this study acknowledge that the FD-Sim scaffold can be a perfect candidate for calvarial defect repair.

Actionable Driver Events in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) stands out as the most aggressive form of lung cancer, characterized by an extremely high proliferation rate and a very poor prognosis, with a 5-year survival rate that falls below 7%. Approximately two-thirds of patients receive their diagnosis when the disease has already reached a metastatic or extensive stage, leaving chemotherapy as the remaining first-line treatment option. Other than the recent advances in immunotherapy, which have shown moderate results, SCLC patients cannot yet benefit from any approved targeted therapy, meaning that this cancer remains treated as a uniform entity, disregarding intra- or inter-tumoral heterogeneity. Continuous efforts and technological improvements have enabled the identification of new potential targets that could be used to implement novel therapeutic strategies. In this review, we provide an overview of the most recent approaches for SCLC treatment, providing an extensive compilation of the targeted therapies that are currently under clinical evaluation and inhibitor molecules with promising results in vitro and in vivo.

Brain-targeted delivery of CRISPR/Cas9 mediated glioblastoma therapeutics

Glioblastoma multiforme (GBM) is a life-threatening malignant tumor of the central nervous system, for which there is currently no effective treatment. Its low survival rate has been interpreted as a result of its high proliferation rate, resistance to apoptosis, and the ability to create a microenvironment conducive to tumor growth. Recently, a powerful and accurate gene-editing tool, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 (CRISPR/Cas9), has high potential in various scientific fields. Such technologies can manipulate cellular mechanisms and defective genes that lead to the progression of many serious diseases such as cancer. One of the major barriers to the application of this technique is the development of a delivery method to diffuse CRISPR/Cas9 efficiently and accurately to the target location in brain. Most existing delivery methods are failed to be translated into clinical result due to the lack of promising safety and efficiency. Thus, I will introduce several strategies here that can be used potentially for CRISPR-Cas9 system delivery in GBM treatment including the principle, advantages, limitations, and latest developments of these systems. This review is composed to provide a concise summary for future researchers to understand the current challenges and approaches in CRISPR/Cas9 mediated GBM therapeutics delivery.

Targeting class A GPCRs for hard tissue regeneration

G protein-coupled receptors (GPCRs) play important roles in various pathogeneses and physiological regulations. Owing to their functional diversity, GPCRs are considered one of the primary pharmaceutical targets. However, drugs targeting GPCRs have not been developed yet to regenerate hard tissues such as teeth and bones. Mesenchymal stromal cells (MSCs) have high proliferation and multi-lineage differentiation potential, which are essential for hard tissue regeneration. Here, we present a strategy for targeting class A GPCRs for hard tissue regeneration by promoting the differentiation of endogenous MSCs into osteogenic and odontogenic progenitor cells. Through in vitro screening targeted at class A GPCRs, we identified six target receptors (LPAR1, F2R, F2RL1, F2RL2, S1PR1, and ADORA2A) and candidate drugs with potent biomineralization effects. Through a combination of profiling whole transcriptome and accessible chromatin regions, we identified that p53 acts as a key transcriptional activator of genes that modulate the biomineralization process. Moreover, the therapeutic potential of class A GPCR-targeting drugs was demonstrated in tooth pulpotomy and calvarial defect models. The selected drugs revealed potent regenerative effects in both tooth and bone defects, represented by newly formed highly mineralized regions. Consequently, this study provides translational evidence for a new regenerative strategy for damaged hard tissue.

A Bioinspired Astrocyte-Derived Coating Promotes the In Vitro Proliferation of Human Neural Stem Cells While Maintaining Their Stemness.

The repair of neuronal tissue is a challenging process due to the limited proliferative capacity of neurons. Neural stem cells (NSCs) can aid in the regeneration process of neural tissue due to their high proliferation potential and capacity to differentiate into neurons. The therapeutic potential of these cells can only be achieved if sufficient cells are obtained without losing their differentiation potential. Toward this end, an astrocyte-derived coating (HAc) was evaluated as a promising substrate to promote the proliferation of NSCs. Mass spectroscopy and scanning electron microscopy were used to characterize the HAc. The proliferation rate and the expression of stemness and differentiation markers in NSCs cultured on the HAc were evaluated and compared to the responses of these cells to commonly used coating materials including Poly-L-Ornithine (PLO), and a Human Induced Pluripotent Stem Cell (HiPSC)-based coating. The use of the HAc promotes the in vitro cell growth of NSCs. The expression of the stemness markers Sox2 and Nestin, and the differentiation marker DCX in the HAc group was akin to the expression of these markers in the controls. In summary, HAc supported the proliferation of NSCs while maintaining their stemness and neural differentiation potential.

Investigation of the Presence of Stem Cells in Rat Uterus Tissue in Postnatal Development Periods

Uterine tissue is an organ with a high proliferation capacity where regeneration, differentiation and shedding are seen. Although this tissue is regenerated every month, the source of it hasn’t been determined yet. Studies are carried out on stem cells being a source that can provide regeneration of the endometrium and the markers expressed by these cells. The purpose of this study was to examine the immunreactivity of CD9 and CD13 expressed by essential population of uterus and CD34, marker of hematopoietic stem cells, at various stages of the postnatal developing pro- cess. In this study, was used 42 female Wistar-albino rats split into six groups; Group I; newborn (2-days), Group II; pubertal (38-days), and Group III: fertile group (12 weeks), by identifying the stages of the estrous cycle in rats a) pro- estrus, b) estrus, c) metestrus, d) diestrus. The expression of CD9, CD13, and CD34 in uterine tissues excised from rats was investigated. In the groups of 38-days and 12-weeks, CD34-expressing cells were present in the stroma of the endometrium next to the myometrium, but such cells were absent in the 2-days group. All groups' uterine epithelium displayed CD9 expression, except for group 2-days. Endometrial stromal cells that expressed CD13 showed only little immureactivity in groups 2 and 38-days, while CD13 expression is noticeable in group 12-weeks. It was thought that the uterine endometrium could be regenerated in puberty and adulthood with the contribution of bone marrow-derived stem cells and uterine-derived epithelial and stromal cells.

Analysis of 9 cases of pediatric-type follicular lymphoma

Objective: To summarize the pathological diagnosis, clinical features, treatment methods and outcomes of pediatric-type follicular lymphoma (PTFL). Methods: Clinical data including the pathology, clinical features, treatment methods, and follow-up results of 9 PTFL patients admitted to Henan Cancer Hospital from February 2017 to February 2023 were analyzed retrospectively. Results: The age of onset in 9 children was 6 to 18 years, all the patients were males. The clinical manifestation was local painless lymph node enlargement in the head and neck, with a stage of Ⅰ-Ⅱ. The histomorphological characteristics of PTFL were similar to those of classic follicular lymphoma (FL). The germinal center of most follicles were enlarged, the mantle zone disappeared, centroblasts were easily visible, and the histological grade were mostly grade Ⅲ, which may be accompanied by the "starry sky" phenomenon. Monoclonal peaks can be seen in B cell clonal rearrangements (BCR). Immunohistochemistry (IHC) showed CD20 positive, CD10 positive, Bcl-6 positive, Bcl-2 negative, C-myc negative, and Ki-67 was 70%-95%. Fluorescence in situ hybridization (FISH) test was negative for t (14, 18), Bcl-2 translocation, and C-myc translocation. Six cases underwent surgical resection, and 3 cases underwent surgical resection combined with chemotherapy. Up to February 2023, with a follow-up time of 45 to 72 months, all children survived without any recurrence and were in a complete remission state. Conclusions: PTFL is mainly characterized by adolescent male onset, with early clinical manifestations and pathological manifestations of high-level histological status, high proliferation index, and lack of t (14; 18)/Bcl-2 translocation and Bcl-2 expression. It is mainly treated by localized surgical excision and has a good prognosis.

Abstract A096: RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma

Abstract Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcoma. Fusion-negative RMS (FN-RMS, the subset lacking FOXO1 translocations), are characterized by oncogenic point mutations in genes that regulate activity of the RAS/ERK signaling pathway. Clinically available agents that directly target oncogenic RAS in cancer have only recently become FDA approved, and their activity is limited to cancers with G12C mutations in KRAS. Alternate molecular strategies to target RAS activation in cancer include inhibitors of RAS regulatory proteins, including the nonreceptor tyrosine phosphatase SHP2. SHP2 regulates RAS signaling by dephosphorylating receptor tyrosine kinases (RTKs), preventing effective RAS GTPase-activating protein (GAP) recruitment. Additionally, SHP2 recruits SOS1, a RAS guanine nucleotide exchange factor (GEF), promoting RAS activation. Novel small molecule inhibitors of SHP2 are now in clinical trials in several cancer types. We set out to determine whether SHP2 inhibitors demonstrate preclinical activity in models of RAS-pathway driven FN-RMS. We hypothesize that specific oncogenic mutations in HRAS, NRAS and KRAS, affecting codons 12, 13 and 61, will determine SHP2 inhibitor sensitivity in models of FN-RMS, and that sensitivity will be determined based on the interactions between RAS and RAS-GAPs/RAS-GEFs. We tested the effects of SHP2 inhibition on ERK pathway signaling, using the small molecule inhibitor TNO155, in a panel of FN-RMS cell lines with varied RAS mutation status. In FN-RMS cells with Q61-mutant HRAS or NRAS, phospho-MEK and phospho-ERK were unchanged, while in cells with RTK-amplification and wild-type RAS, or those with G12 or G13-mutant HRAS we observed reductions in p-MEK and p-ERK following treatment with TNO155. Reduced 2-D and 3-D cell growth were also observed in WT and G12/G13-mutant HRAS cell lines, but not those with Q61 mutations, using both high throughput proliferation assays and soft-agar colony formation assays, respectively. Responses in sensitive cells were modest and cytostatic at best, suggesting single agent SHP2i may have insufficient activity in these tumors. RNAseq analysis of TNO155-sensitive RAS-WT RMS cells reveals a set of differentially expressed genes that may inform the selection of agents for combination therapy. In addition, a subset of FN-RMS cells also harbor activating mutations in FGFR and PI3K-associated genes, and we therefore tested whether TNO155 might synergize with FGFR or PI3K small-molecule inhibitors and found additive effects between TNO155, the PI3Ki alpelisib, and the pan-FGFRi erdafitinib. In conclusion, FN-RMS cells driven by RTK-amplification, or by RAS G12 or G13 mutations, are sensitive to TNO155 while cells driven by RAS Q61 mutations are not. Additionally, FN-RMS cells treated with TNO155 may rely on the PI3K pathway for adaptive resistance, and combination treatment involving TNO155 and FGFR or PI3K inhibitors may reduce tumor growth with better efficacy than either compound alone. Citation Format: Andrew Baker, Patience Odeniyide, Alyza Skaist, Alla Lisok, Elana Fertig, Jiawan Wang, Christine Pratilas. RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma [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 A096.

Reassessing specificity/selectivity of taxane-based chemotherapy

<p class="MsoNormal" style="margin-top: 6pt; line-height: 13pt; text-align: justify;"><span lang="EN-US" style="font-family: arial, helvetica, sans-serif;">The paramount prerequisite for effective anti-cancer drugs is their ability to eradicate malignant cells while sparing non-cancer cells. The divergence in properties between malignant and non-cancer cells establishes a "therapeutic window," a critical consideration for achieving desirable treatment outcomes. Central to this is the imperative of a cancer drug's "selectivity and specificity." Taxanes, a pivotal class of successful anti-cancer drugs, continue to serve as the linchpin of cancer treatment due to their efficacy across a spectrum of cancer types. Operating as broad-spectrum chemotherapeutic agents, taxanes exert cytotoxic effects on proliferative cancer cells by binding to and stabilizing microtubules, disrupting mitosis, inducing mitotic catastrophe, and resulting in cell death. The distinct proliferative nature of cancer cells, as opposed to less proliferative non-cancer cells, affords taxanes a measure of specificity and selectivity. Nevertheless, sporadic yet recurring evidence suggests that taxanes also operate through <span style="color: black; mso-themecolor: text1;">non-mitotic mechanisms. Taxanes' binding and stabilization of microtubules lead to micronucleation and subsequent cell death, impacting both mitotic and non-mitotic cells. Recent discoveries indicate that the flexible and weakened nuclear envelope of malignant cells renders them sensitive to taxane-mediated micronucleation and cell death during various phases of the cell cycle. Conversely, non-cancerous cells typically exhibit a more robust and sturdy nuclear envelope, rendering them more tolerant to taxane-induced nuclear envelope fragmentation and subsequent micronucleation </span>and cell death. The expression levels of nuclear envelope structural proteins, particularly Lamin A/C, emerge as indicators of taxane sensitivity. This evolving understanding underscores that nuclear envelope malleability, in conjunction with a high proliferation rate, is a pivotal determinant of taxane specificity and selectivity against malignant cells. These insights necessitate reconsidering oncological strategies to augment taxane efficacy, overcome resistance, and mitigate side effects.</span></p>

Reassessing specificity/selectivity of taxane-based chemotherapy

<p class="MsoNormal" style="margin-top: 6pt; line-height: 13pt; text-align: justify;"><span lang="EN-US" style="font-family: arial, helvetica, sans-serif;">The paramount prerequisite for effective anti-cancer drugs is their ability to eradicate malignant cells while sparing non-cancer cells. The divergence in properties between malignant and non-cancer cells often establishes a "therapeutic window," a critical consideration for achieving desirable treatment outcomes. Central to this is the imperative of a cancer drug's "selectivity and specificity." Taxanes, a pivotal class of successful anti-cancer drugs, continue to serve as the linchpin of cancer treatment due to their efficacy across a spectrum of cancer types. Operating as broad-spectrum chemotherapeutic agents, taxanes exert cytotoxic effects on proliferative cancer cells by binding to and stabilizing microtubules, disrupting mitosis, inducing mitotic catastrophe, and resulting in cell death. The distinct proliferative nature of cancer cells, as opposed to less proliferative non-cancer cells, affords taxanes a measure of specificity and selectivity. Nevertheless, sporadic yet recurring evidence suggests that taxanes also operate through non-mitotic mechanisms. Taxanes' binding and stabilization of microtubules lead to micronucleation and subsequent cell death, impacting both mitotic and non-mitotic cells. Recent discoveries indicate that the flexible nuclear envelope of malignant cells renders them sensitive to taxane-mediated micronucleation and cell death during various phases of the cell cycle. Conversely, non-cancerous cells typically exhibit a more robust nuclear envelope, rendering them more tolerant to taxane-induced nuclear envelope fragmentation and subsequent micronucleation and cell death. The expression levels of nuclear envelope structural proteins, particularly Lamin A/C, emerge as indicators of taxane sensitivity. This evolving understanding underscores that nuclear envelope malleability, in conjunction with a high proliferation rate, is a pivotal determinant of taxane specificity and selectivity against malignant cells. These insights necessitate reconsidering oncological strategies to augment taxane efficacy, overcome resistance, and mitigate side effects.</span></p>

Genomic and Transcriptomic Landscape of Hyper-Apobec Multiple Myeloma

Introduction APOBEC mutational signatures are one of the most important mutational signatures in newly diagnosed multiple myeloma (NDMM). APOBEC mutagenesis primarily works through two enzymes, APOBEC3A (A3A) and APOBEC3B (A3B). While these genes are expressed in most NDMM patients, their influence on APOBEC mutagenesis appear to be extremely heterogeneous, with some patients having no detectable mutations and others having high contribution (hyper-APOBEC). Methods To decipher the mechanism of APOBEC mutagenesis, we interrogated 752 low coverage long insert whole genomes, 723 whole exomes (WES) and 767 RNA sequencing (RNA-Seq) from NDMM patients enrolled in the CoMMpass study. Results APOBEC mutational activity was identified in 416/723 (57.5%) patients using WES. Of these, 41 (5.8%) had MAF/MAFB translocations. Overall, 48/723 (6.6%) patients were defined as hyper-APOBEC (13 without MAF/MAFB events). We used differential expression (DE) analysis between three groups: hyper-APOBEC with MAF/MAFB (HA_TRA: n=31), hyper-APOBEC without MAF/MAFB (HA_NORM: n=8), and non-hyper-APOBEC without MAF/MAFB (WT: n=510). 534 genes were significantly associated with hyper-APOBEC independently from MAF/MAFB events. Importantly, A3A and A3B expression were higher in hyper-APOBEC compared to other cases (logFC=2.38, logFC=1.58 respectively). Using Spearman's correlation, we identified 50 significantly correlated genes (r-squared>0.18; p<0.01) with A3B. Most of the genes belonged to the APOBEC-associated group defined by the DE and were enriched for cell cycle/proliferation activity, suggesting a link between highly proliferative disease and hyper-APOBEC. To validate this finding, we evaluated the link between these APOBEC-associated genes and hyper-APOBEC among ICGC breast cancers, a tumor known to have high prevalence of APOBEC mutagenesis. Overall, 35/50 of these genes showed similar correlation between hyper-APOBEC breasts and MM. Interestingly, most of these genes appear to be negatively controlled by E2F4 (DREAM complex), and positively controlled by E2F1, FOXM1, and MYBL2 (i.e. cell cycle genes). In line with mouse models (Roelofs et al., eLife, 2020), our data confirmed that highly proliferating tumors suppress the DREAM complex leading to induction of A3B. Overall, these data suggest a strong link between proliferation and A3B expression and mutagenesis. However, not all high proliferating tumors had high APOBEC mutagenesis. To better investigate this aspect, we compared 139 known MM genomic events to identify potential differences between the hyper-APOBEC group (HA) and the rest of the cohort (WT). HA_TRA and HA_NORM showed similarities, including a highly complex genomic profile with enrichment for 1q gain/amp (p < 0.02), 13q (p < 0.05) and 16q deletions (p<0.01). Interestingly, 5/13 (38.4%) of HA_NORM samples harbor CCND1 translocation. In terms of gene expression, both groups showed enrichment for GEP70 indicating a high-risk transcriptional profile apart from MAF/MAFB overexpression. Finally, HA_TRA and HA_NORM showed similar poor outcomes compared to WT. To further validate the association between APOBEC and complex genomic profile, we explored again the ICGC breast WGS, observing a strong correlation between homologous recombination deficiency (HRD) and hyper-APOBEC (p < 0.05). To validate the relationship between the DREAM complex and APOBEC, we pharmacologically inhibited FOXM1 (FDI6, 20uM) and E2F4/6 (HLM00074, 20uM) in 8226, U266 and MM1.S multiple myeloma cell lines and showed increased A3B levels upon 72h of E2F4/6 inhibition. Simultaneously, FOXM1 inhibition confirmed A3B downregulation. Conclusion Overall, our data support a model where, APOBEC genes are variably expressed in all NDMM, but only patients with high proliferation rate and high level of complexity acquire mutations (Figure 1).

Single-Cell RNA-Seq Reveals Intermediate Cell States and Identifies Features Defining Cellular Heterogeneity in Inv(16) Acute Myeloid Leukemia (AML)

AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. One recurrent chromosomal translocation common in AML is chromosome 16 inversion, inv(16)(p13.1q22), which results in the leukemogenic fusion gene CBFB- MYH11. Using a conditional Cbfb-MYH11 (CM) knock-in mouse model to mimic the somatic expression of the CM fusion gene, we demonstrated that CM expression leads to impaired hematopoietic differentiation as well as accumulation of phenotypic hematopoietic stem cells (HSC) and pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitors, which are predisposed to leukemic transformation. Flow cytometry-based phenotypic analysis revealed that the transformed leukemic blasts are predominantly characterized as pre-granulocyte-macrophage progenitor (pre-GM) (Lin −cKit +Sca1 −CD16/32 -/loCD150 −CD105 −). However, cell type characterization via conventional surface markers is limited by known cell markers and is incapable of resolving pathologic heterogeneities. We previously reported that modeling AML in a continuum of differentiation states using single-cell RNA-seq (scRNA-seq) data predicts intermediate states of differentiation in AML. Herein, we utilized scRNA-seq to characterize CM leukemic blasts, and identified transcriptomic alternations and features that defined the leukemic cell states and heterogeneities. CM expression was induced by poly (I:C) and mice (n=3) were monitored for leukemia progression. At the leukemic end-stage (detection of >50% of circulating cKit + cells), we collected unfractionated and ckit + sorted peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells. Similarly poly(I:C)More than 105,000 cells harvested from the WT and CM leukemic mice were analyzed by scRNA-seq. We applied uniform manifold approximation and projection (UMAP) for dimensionality reduction and performed cell type annotation by SingleR. First, we mapped mature hematopoietic cell types in the WT PB and hematopoietic stem/progenitor cell populations (HSPC) in the WT BM, and in contrast, leukemic PB and BM were highly similar and showed distinct cell clusters compared to WT. To characterize HSPC subpopulations, we focused on cKit+ sorted WT and leukemic BM cells, visualized them via UMAP, and performed clustering and cell type annotation. The leukemic cKit+ cells were mostly identified as megakaryocytic-erythroid progenitor (MEP)-like HSC, revealing a discrepancy between flow cytometry-based phenotypic characterization and transcriptome-based cell type assignment. These leukemic MEP-like HSCs showed up-regulation of erythroid differentiation genes, including Hba-a1, Hba-a2, Hbb-bs, Hbb-bt and Sox6, together with decreased expression of granulocytic markers such as Elane, Prtn3, S100a8, S100a9 and Mpo. Comparison of leukemic MEP-like HSCs vs WT MEP clusters via gene set enrichment analysis (GSEA) with a threshold of nominal p-value ≤ 0.05 and FDR ≤ 0.05 in 50 hallmark gene sets (MSigDB) indicated dysregulated pathways, including up-regulation of “TNFα signaling pathway via NFκB”, “inflammatory response”, and “UV responsive downregulated genes”; and down-regulation of “MYC target V1”, “MYC target V2”, “E2F targets”, “G2M checkpoint”, and “oxidative phosphorylation”. Similarly, GSEA in Gene Ontology (GO) showed downregulation of pathways relevant to mitochondrial protein translation and ribosomal biogenesis/assembly in leukemic MEP-like HSCs, compared to WT MEP. In addition, cKit+ BM cells can be separated into 31 clusters (C0-C30), of which 9 leukemic clusters were identified. Through gene expression-based cell cycle scoring, single sample gene set enrichment (ssGSEA), and marker identification, these 9 leukemic clusters can be defined by several features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). Altogether, these data reveal intermediate differentiation cell states in inv(16) leukemic cells which are highly heterogeneous populations with distinct cell cycle stages, metabolic activities, and marker expression.

A Novel Strategy to Produce CAR-Γδ T Cells Via Targeted Knockout By CRISPR/Cas9 and Targeted Knockin By AAV

Chimeric antigen receptor (CAR)-γδ T cells are promising in immunotherapy owing to the dual recognition system of γδ T cells, which is potential to exhibit stronger anti-tumor effect. Up to date, established CAR-γδ T cells exhibit poor anti-tumor efficiency and are commonly generated using lentiviral (LV) or gamma-retroviral (γRV) vectors, which may increase the risk of insertional mutagenesis. Nowadays, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used to cleave the genomic sequence precisely, as well as Adeno-associated Virus (AAV) being used to deliver DNA templates for target sequence knock-in (KI) mediated by homology-directed repair, which could contribute to achieve the accurate insertion of CAR at a specific site. Furthermore, the endogenous promoter of TCR has been used to express CAR molecules with stable expression and better antitumor function in the production of CAR-αβ T cells. In the present study, we established and optimized the strategy to generate CAR-γδ T cells, combining the site-specific knock-out (KO) targeting the TCR delta chain constant region (TRDC) site of γδ T cells using CRISPR/Cas9 and site-specific CAR knock-in using AAV. Firstly, we screened out a functional guide RNA (gRNA) targeting the γδ TCR TRDC locus by prediction tools and validation in cell line model experiments, and achieved an INDEL frequency of 31.8%, which showed a high activity of gRNA. Then, we optimized the option to increase KO efficiency including exploring appropriate Cas9 amount, ratio of gRNA to Cas9 and cell number titration. Besides, we designed AAV vector carrying CAR sequence targeting the gRNA aiming site selected above (Figure1A). After that, we used CRISPR/Cas9 and AAV to knock out TCR and knock in CAR sequence in γδ T cells. We tried different electroporation procedures (X001, T007, U014, T023) and cell culture conditions (cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days or for 10 days) to select out the optimal procedure to acquire high KO/KI efficiency and high cell proliferation rate. When we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days (Day 5) to generate CAR-γδ T cells, cells electroporated using X001 procedure showed the best TCR KO efficiency of ~80%（p<0.05）and CAR expression of ~20% (p< 0.01), as well as keeping the highest cell proliferation rate than other procedures (p<0.0001) (Figure 1B and 1C). If we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 10 days (Day 10) to generate CAR-γδ T cells, U014 program also exhibited a relative high CAR expression and the significant quicker cell growth than others in the later period (p<0.01) (Figure 1B and 1C). However, the later proliferation rate of CAR-γδ T cells generated at Day 5 was superior to that generated at Day10. Thus, we determined that a 5 days' cell culture and the use of X001 program were the optimal condition to generate CAR-γδ T cells. Besides, we found that KI efficiency is positively correlative with Multiplicity of infection (MOI). While MOI comes to 1.2×10 5, the KI efficiency couldn't be increased (Figure 1D). Finally, we evaluated the anti-tumor function of CAR-γδ T cells . Antigen stimulated CAR-γδ T cells significantly released TNFα and INFγ, but failed to produce IL2 (Figure 1E). Besides, CAR-γδ T cells could significantly lysis different tumor cells even at a low effector/target ratio (E/T) (Figure 1G). Interestingly, the proportion of memory phenotype (CD62L + ) in CAR-γδ T cells after being stimulated was comparable to that of CAR-γδ T cells without stimulation, indicating the potential of the great anti-tumor activity of CAR-γδ T cells after being stimulated in tumor environment (Figure 1F). Our new method successfully generated CAR-γδ T cells with a CAR expression of 20% and in a high proliferation rate, which was determined to exert effective anti-tumor effects.

First Report of Asymptomatic Solitary High-Grade B-Cell Lymphoma NOS: An Enigmatic Entity

Background High-grade B cell lymphoma (HGBL) is a new disease entity introduced by WHO in 2016, including two types: HGBL with a double hit (DH) or triple hit (TH) and HGBL, not otherwise specified (NOS). HGBL DH/TH are defined by genetic rearrangements of specific oncogenes MYC, BCL2 and/or BCL6, while HGBL NOS is defined purely base on morphology in the absence of these genes. HGBL DH/TH is known for its poor prognostication given its propensity to rapidly progress and disseminate, however HGBL NOS remains ill-defined and poorly understood with no tangible literature describing its clinical nature and therapeutic susceptibility. Case presentation A 76-year-old post-menopausal woman with no significant PMH presented for evaluation of painless hematuria during a routine office visit. Evaluation with imaging showed a suspicious incidental liver mass concerning for possible metastatic or primary liver cancer. Obtained labs were unremarkable, including serum LDH, serological testing for hepatitis B/C and HIV1/2, and typical tumor markers. PET scan showed focal uptake of tracer in liver with no nodal involvement. Liver biopsy revealed diffuse infiltration by atypical lymphocytes. Immunohistochemical analysis showed CD20 and CD10 positivity, negative CD30, and high Ki-67 proliferation rate of 100%, however the absence of MYC, BCL6, BCL2, and t(8;14) rearrangements on FISH ruled out Burkitt and DH/TH lymphoma, consistent with HGBL-NOS. Following discussions with Henry Ford Lymphoma Tumor Board, patient was treated with six cycles of R-CHOP, where follow-up imaging indicated remission of her lymphoma. Conclusion This represents an extraordinary first report of HGBL NOS manifesting as an isolated primary hepatic lymphoma in the complete absence of clinical symptoms. The significance of this case lies in its atypical extra-nodal site of involvement and total absence of clinical symptoms and serological markers, underscoring the unfulfilled need to further describe this enigmatic entity.

JAK2 Kinase Fusion and Myeloid Involvement in Acute Lymphoblastic Leukemia

Introduction JAK2 fusion genes (FGs) are rare but can be found in various phenotypes of myeloid or lymphoid malignancies. Both in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC, PMID: 35767897), JAK2-FGs were included in the diagnostic entity of myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK or MLN-eo), and both suggest MLN-TK/eo as a priority diagnostic entity. It is highlighted in ICC that cases presenting as acute lymphoblastic leukemia (ALL) with M/LN-eo differ from de novo B-ALL and T-ALL in the involvement of myeloid cells. There is still a lack of research reports about how many JAK2-FG-positive ALL cases developed from M/LN-eo or had myeloid involvement. Methods A total of 21 ALL cases (Table 1) were enrolled in this study. The positive for JAK2-FGs were determined by G-banding karyotype analysis, transcriptome sequencing (RNA-seq; PMID: 34135310), reverse transcription PCR (RT-PCR), and interphase fluorescence in situ hybridization (I-FISH) with the JAK2 break-apart probe. Gene mutation analysis (PMID: 29932212) was also performed. According to whether there were FISH-JAK2 split signals in rod and lobed nuclear cells (Figure 1A), the cases were divided into My+ group (with myeloid involved) and My- group (without myeloid involved), respectively. Results Among these cases, there were 14 males and 7 females (M:F = 2:1); 15 B-ALL and 6 T-ALL cases; the median age was 22 years old (range 5-55). All cases were positive for JAK2-FGs in RNA-seq analysis, and all FGs were verified by RT-PCR and Sanger sequencing. A total of 16 JAK2 partner genes were detected (Table 1, Figure 1B/C), of which FNBP1, CDK5RAP2, GTF2I, and C14orf 93 have not been reported previously. All cases retained the complete kinase domain of the JAK2 gene, and 13 cases retained part or all of the JAK2 pseudo-kinase domain in their JAK2-FGs. Karyotype abnormalities were detected in 17 cases (81%), with 9p24 involved in 10 cases (48%), abnormalities other than 9p24 were observed in 7 cases, and karyotype was normal in 4 patients. FISH-JAK2 inspection showed split signals in 20 cases and showed negative results in case #21 who with a complex karyotype abnormality. Twelve cases (12/20=60%) showed JAK2 split signals in rotundum, rod, and lobed nuclei (Table 1, My+ group), suggesting both lymphoid and myeloid lineages were involved. We further investigated samples of 4 cases at the time of morphological complete remission after induction chemotherapy, of which the immunophenotype analysis was also negative for lymphoid blast cells. FISH-JAK2 inspection showed split signals accounting for 13%~79% of the nucleus, with split signals in the rod/lobed nucleus, and RT-PCR also confirmed abundant JAK2-FG transcripts in these samples. There were 8 B-ALL and 4 T-ALL cases in the My+ group, of which 2 T-ALL cases were also positive for STIL::TAL1 fusion, and 3 cases experienced eosinophilia during the course of the disease (Table 1). The 5 cases whose JAK2 partner genes were lymphoid development-related transcription factors (ETV6, PAX5) were all in the My- group. All My- cases and #21 had no documented eosinophilia. Among the 21 cases in this cohort, 13 had white blood cell (WBC) counts >50×10 9/L at diagnosis, and no significant hepatosplenomegaly. The total follow-up time was 2-89m, with a median overall survival (OS) time of 17m. Fourteen cases underwent allo-HSCT, and the median OS was 29.5m in the HSCT group and 9m in the non-HSCT group, respectively. The median OS was 15.5m in the My+ group and 29m in the My- group. The leukemia cells in these cases are prone to be in a high and rapid proliferation state, and they generally have a short OS and dismal prognosis. Conclusions Myeloid involvement accounted for 60% of JAK2-FG positive ALL and My+ cases had a worse prognosis and shorter OS than My- cases in this study. I-FISH inspection can be used as a method to determine whether there was myeloid involvement and provide valuable information for accurate analysis. The incidence of JAK2-FGs is low in ALL, but the fusion partners are diverse. This group of cases is more likely to have cryptic JAK2 translocations and with dismal prognosis. The phenotype of JAK2-FG malignancies is jointly determined by multiple factors under the influence of partner genes, accompanying fusion genes, and accompanying mutations.

The Immune Editing Process Reshapes the AML1-ETO Fusion Protein-Induced Pre-Leukemia Microenvironment

Objective: Currently, the process of common chromosome translocation, such as AML1-ETO, recruiting chromatin-modifying enzymes that drive the malignant phenotype is an object of intense investigation. However, there exists an indeterminate understanding of how this chromosome translocation reshapes the immune microenvironment, and whether immune editing occurs during the pre-leukemia stage to facilitate their own progression. In this study, we evaluate how the AML1-ETO chromosome translocation reshapes the immune microenvironment by employing the Aml1 Eto/+; Mx1-Cre mouse model to identify potential pivotal immunotherapy targets. Methods: Four weeks after PIPC induction (5mg/kg), bone marrow-derived T cells without the AML1-ETO (AE) fusion gene and Lin - c-Kit + Sca-1 + (LSK) cells were sorted from both Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group), respectively, and subsequently mixed at 8:2 ratio for single-cell RNA-Seq(scRNA) and single-cell TCR-seq. The results are further validated by real-time PCR and flow cytometry. Results: In our preliminary cell-sorting-based dissection research, we found that despite antigen presentation existing in clonally expanded AE precursor cells and the differentiated T cells like effector T cells expanded, the established AE-expressed precursor cells are not eradicated by the host immune system as expected. To describe the mechanism underlying it, the first step is to find whether the potentially tumor-reactive T cells (pTRTs) existed. We examined the single-cell data based on the following four dimensions: the tumor-enrichment index, the proliferation index, the clonal expansion index, and the TCR signaling pathway activation index. Two clusters of Cd8 + exhausted T cells and four clusters of Cd4 +Runx3 + cytotoxic T cells were identified as pTRTs. While Cd8 + pTRTs demonstrated high expression levels of genes like Gzmk, Ifng, and Prf1, they also exhibited high expression levels of exhaustion markers such as Pdcd1, Tox, and Ctla4. More importantly, compared with virus-specific T cells recognized by machine learning trained from validated TCR databases, the Cd8 + pTRTs showed the feature of metabolic insufficiency via the Compass analysis. Cell-cell interaction analysis by CellphoneDB and NicheNet and following flow cytometry confirmation revealed that the highly expressed PVR and NECTIN2 on AE + precursor cells interacted with pTRTs via inhibitory receptor TIGIT, while they interacted with bystander T cells through stimulatory receptor CD226. At the same time, Tregs played a vital role in the maintenance of the immune equilibrium state as the Treg cells increased in the AE microenvironment but not homogeneously. There were four different Treg clusters identified, including Cd4_Treg_Ccr7+, Cd4_Treg_Tnfrsf9-, Cd4_Treg_ Cdc25b+ with high apoptosis and proliferation score, and Cd4_Treg_Tnfrsf9+ with high suppression\exhaustion/activation/migration score which was recognized as effector Treg. It was observed that only the effector Treg exhibited an increase in proportion. Monocle analysis showed that Cd4_Treg_Ccr7+ was at the beginning of common origin while the Cd4_Treg_Cdc25b+ and Cd4_Treg_Tnfrsf9+ were at the endpoint of two distinct differentiation pathways and the AE microenvironment shifted to the latter one. The flow cytometry results clearly showed a notable rise in KI67 -TNFRSF9 + effector Tregs, along with a significant decline in KI67 +TNFRSF9 - proliferative Tregs. Conclusions: Our findings demonstrate that, during the pre-leukemia stage, the immune editing process has already occurred, leading to substantial alterations in the immune microenvironment. It was identified that PVR/NECTIN2-TIGIT interaction and TNFRSF9 +Treg as targetable mechanisms to restore the function of pTRTs and break the immune equilibrium dilemma.

Lisocabtagene Maraleucel (liso-cel) in Patients (Pts) with R/R MCL: Subgroup Analyses in Pts with High-Risk Disease Features from the MCL Cohort of the TRANSCEND NHL 001 Study

Background: Pts with R/R MCL after ≥ 2 prior lines of therapy, including a Bruton tyrosine kinase inhibitor (BTKi), have poor prognosis. Among pts with R/R MCL, several high-risk disease features are associated with a worse prognosis, including TP53 mutation, high proliferation index (Ki-67 ≥ 30%), blastoid morphology, and secondary CNS involvement. Liso-cel is an autologous, CD19-directed, 4-1BB CAR T cell product administered at equal target doses of CD8 + and CD4 + CAR + T cells. In the primary analysis of the MCL cohort from the phase 1, seamless design TRANSCEND NHL 001 study (NCT02631044), liso-cel treatment resulted in a rapid, high rate of durable CRs with a manageable safety profile in pts with heavily pretreated R/R MCL. Here we report the outcomes in pts with R/R MCL from prespecified subgroup analyses based on high-risk disease features. Methods: Eligible pts had PET-positive R/R MCL after ≥ 2 lines of prior therapy, including a BTKi, alkylating agent, and CD20-targeted agent. Pts received liso-cel at a target dose of 50 × 10 6 or 100 × 10 6 CAR + T cells after lymphodepleting chemotherapy. Bridging therapy was allowed. Primary endpoints were treatment-emergent AEs (TEAE) and ORR by independent review committee (IRC) per Lugano 2014 criteria; secondary endpoints included CR rate, duration of response (DOR), PFS, and OS. The liso-cel-treated set included all pts who received liso-cel. The efficacy analysis set included all pts in the liso-cel-treated set who had PET-positive disease per IRC at baseline. Results: Of 104 leukapheresed pts, 88 received liso-cel. Median on-study follow-up was 16.1 mo (range, 0.4-60.5). Median age was 68.5 y (range, 36-86) and median prior systemic lines of therapy was 3 (range, 1-11). Among liso-cel-treated pts, 66 (75%) had Ki-67 ≥ 30% and 15 (17%) had Ki-67 < 30% (Ki-67 was not reported in 7 [8%] pts); 20 (23%) had a TP53 mutation present and 34 (39%) did not ( TP53 mutation was indeterminate in 4 [5%] pts and not reported in 30 [34%] pts); and 27 (31%) had blastoid morphology and 48 (55%) did not (blastoid morphology was not reported in 13 [15%] pts). Most pts with these high-risk disease features also had disease that was refractory to last therapy, was refractory to prior BTKi, and had a complex karyotype. A total of 7 (8%) pts had secondary CNS lymphoma, of which 5 had refractory disease, 5 had Ki-67 ≥ 30%, and 1 each had TP53 mutation and blastoid morphology. Response rates, PFS, and OS across subgroups were consistent with the overall population, with high ORR and CR rate that was durable and high PFS and OS (Table 1). Although a limited number of pts had TP53 mutation assessed, pts with TP53 mutation had a numerically lower median DOR than the overall population, likely due to a higher proportion of objective responders achieving a PR than CR; however, pts with TP53 mutation who achieved CR had durable responses with 6 of 11 pts in an ongoing response at data cutoff. Among the 7 pts with secondary CNS lymphoma, response rates were high (ORR, 86% [n = 6]; CR rate, 71% [n = 5]), and 3 of 5 pts who achieved CR were in an ongoing response at data cutoff. Safety outcomes across subgroups were generally consistent with the overall population (Table 2). Most cytokine release syndrome (CRS) and neurological events (NE) were low grade in all subgroups, similar to the overall population (any-grade CRS, 61% [grade 3-4, 1%]; any-grade NEs, 31% [grade 3-4, 9%]; and no grade 5 CRS or NEs). Incidences of other TEAEs of special interest were also similar among subgroups. Of the 7 pts with secondary CNS lymphoma, 5 had low-grade CRS and 3 had low-grade NEs with no grade ≥ 3 CRS or NEs reported; 1 pt had a grade ≥ 3 infection. Cellular kinetics and B-cell aplasia by subgroup will be presented. Conclusions: In these subgroup analyses including pts with R/R MCL and high-risk disease features (Ki-67 proliferation index ≥ 30%, TP53 mutation, blastoid morphology, and secondary CNS lymphoma), liso-cel demonstrated clinically meaningful efficacy across subgroups with durable responses. Efficacy and safety outcomes were generally consistent with the overall study population. While some subgroups were limited by small numbers, these results suggest a favorable benefit/risk profile for liso-cel in pts with R/R MCL and high-risk disease features, a pt population for whom effective treatment options are rarely available.

Hspa5 Deficiency Blocks Intestinal Enterocyte Differentiation in Graft-Versus-Host Disease

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective therapy for hematological malignancies. Graft-versus-host disease (GVHD) is a life-threatening complication, and the intestine is one of the major sites affected. Currently, immunosuppressive agents remain the preferred choice for GVHD treatment. However, treatment resistance still occurs in a large population, indicating that some tissue damage may be irreversible. Methods: The murine transplant and GVHD model were established as previously described. To characterize the intestinal damage mediated by radiation and GVHD, murine small intestine tissues were collected at day 14 after transplantation. Primary small intestinal crypts were isolated from each group and cultured for five consecutive days to evaluate their ability to proliferate and differentiate. Single-Cell analysis was performed to characterize the impaired intestinal repair after injury. Results: At day 14, the villus-to-crypt ratio was partially recovered in the BM group, but remained imbalanced in the GVHD group. We observed a significant decrease in the expression of Reg1, a gene expressed by enterocytes (EC), in the GVHD groups compared to the BM group. we further utilized a 3D organoid model to investigate intestinal growth and differentiation. The organoid size from the GVHD group was comparable to the BM and control groups. However, the number of buds per organoid was significantly reduced in the GVHD group compared to the BM and control groups. Immunofluorescence staining confirmed that cells in GVHD-derived organoids exhibited high proliferation (stained by Mki67) but impaired differentiation of enterocyte (stained by Aldob). To gain insights into the cellular mechanisms influencing enterocyte differentiation, we dissociated murine small intestinal tissues from each group into single cells and sequenced them using the 10X Genomics platform. Through pseudochronological analysis, each cell was ordered along the predicted lineages and assigned a pseudotime. We found that enterocytes in GVHD exhibited a long and discontinuous trajectory, suggesting explicit blockage of EC differentiation. Functional enrichment analysis showed that pathways related to cell growth and differentiation, such as MAPK, ERBB, and inositol phosphate metabolism, were highly enriched in M2 of the control and BM groups. In contrast, MAPK was dramatically downregulated at the early stage of ISC differentiation in the GVHD group. Next, we analyzed the differentially expressed genes in EP between the GVHD and BM groups. Endoplasmic reticulum (ER) chaperone-related genes from the heat shock protein (Hsp) 70 family (Hspa1a/1b/5), Hsp90 family (Hsp90aa1/ab1), and Hsp40 family (Dnajb1) were significantly decreased in GVHD compared to BM. Notably, Hspa5 (also known as GRP78 or Bip), which serves as a sensor of glucose levels and misfolded proteins, exhibited reduced protein levels, along with decreased IRE1ɑ phosphorylation (p-IRE1ɑ) and Xbp1s, as well as decreased PERK phosphorylation (p-PERK) and ATF6 expression. To validate our conjecture, we utilized small intestinal organoids cultured for 3 days for further experimentation. RT-qPCR demonstrated that energy restriction downregulated the mRNA expression of Hspa5 and other chaperones. Furthermore, we treated intestinal organoids with HA15, a BIP inhibitor, which resulted in significantly enlarged area but fewer buds. Consistent with our previous findings, Hspa5 was downregulated in the HA15-treated group, along with decreased expression of Hsp family and enterocyte markers. Since Hsp family plays key roles in restoring cellular homeostasis, we wondered if upregulating Hspa5 could protect intestinal cells from outside stimuli such as pro-inflammatory cytokines. When intestinal organoids were cocultured with IFN-g, we found that Bix, the selective Bip inducer, protected organoids from apoptosis by increasing Hsp70 expression. Conclusion: Given that immunosuppression partly resolves GVHD, strategies aimed at restoring intestinal homeostasis should be given sufficient attention. Our study reveals that intestinal enterocyte differentiation is blocked in GVHD, possibly due to starvation and energy restriction. Deficiency in Hspa5 results in impaired intestinal repair, and increasing Hsp levels may enhance cell resistance to cytokine storms.

Remodeling of the Immune Microenvironment By Oncogenic MYD88 Dictates Immunotherapy Responses across Indolent and Aggressive B-Cell Lymphomas

The MYD88 L265P mutation is detected in a variety of lymphoma subtypes, ranging from indolent MALT or lymphoplasmacytic lymphoma (LPL) to aggressive DLBCL with activated B-cell (ABC) phenotype. Whether oncogenic MYD88 L265P constitutes a common founder mutation and how it drives lymphomas with different clinical, histopathological, and immunological characteristics, remains unknown. To address this fundamental question, we screened mice genetically engineered to express an orthologous MYD88 L252P mutation crossed with animals carrying secondary genetic lesions common in MYD88 L265P lymphomas, triggered at two B-cell differentiation stages. Several mouse strains developed indolent or aggressive lymphomas depending on the cell of origin and the secondary genetic lesion. In two of the models, human-like MALT or LPL were progressively driven by mutant MYD88 activated from immature pre-B lymphocytes with either constitutive BCR signaling or BCL2 expression. High-throughput cellular and molecular characterization of the tumor immune microenvironment (TME) by single-cell RNA sequencing coupled with BCR/TCRseq revealed bi-directional B-cell interactions with CD4 + Tfh and Th1 cells from early disease state, promoting survival of B-cell clones through CD40L:CD40 signaling. During progression, IL10-secreting regulatory T (Treg) cells were recruited into the tumor niche through a Ccl5-Cxcr3 axis to suppress CD8 + T-cell cytotoxicity. In patients with MYD88 L265P LPL characterized at progressive disease states, CD40:CD40L interaction between tumor and immune cells took place early while Tregs accumulated at late stage. In the LPL model and in human MYD88 L265P-LPL cells, CD40 signaling decreased effects of standard-of-care drugs including BTK inhibitors. Accordingly, anti-CD40 therapy disrupted B/T-cell interactions and increased ibrutinib activity. In conclusion, oncogenic MYD88 triggered at early B-cell stages promotes indolent lymphomas that are sustained by CD40 signaling, which restricts therapy responses that can be restored upon CD40 blockade. When MYD88 L265P was activated in mature germinal-center B cells together with either Prdm1 or Trp53 deletion, mice developed extranodal ABC-DLBCL (MCD/C5 genomic class). IHC, flow cytometry and single-cell RNAseq revealed that DLBCL with Prdm1 loss showed morphologically heterogeneous B cells with abundant immune and T cells with activated/exhausted phenotypes, which recapitulated the inflammatory/immunosuppressive TME category in DLBCL patients (IN-LME, Kotlov- Cancer-Discovery-2021). In contrast, DLBCL with Tp53 deletion exhibited monotonous B-cell infiltrates with high proliferation index and a TME with depletion of T cells with effector/memory phenotypes and CD4 +CD25 +Foxp3 + Tregs, consistent with the immune depleted TME category (DP-LME). Analysis of 1,039 DLBCL patients demonstrated that majority of MCD cases with MYD88 L265P showed either an IN-LME (43%) or DP-LME (44%). Consistent with the models, ABC-DLBCL patients with MYD88 L265P and TP53 loss/mutation exhibited highly proliferative disease with a DP-LME, while ABC-DLBCLs with MYD88 L265P but without TP53 abnormalities showed an IN-LME. Notably, the number of infiltrating CD4 + T cells correlated with TP53 activity score (PROGeNY) across MCD-DLBCLs. Overall, the presence or absence of TP53 alterations in MYD88 L265P ABC-DLBCL induces TME characterized as DP-LME or IN-LME, respectively. Since DP-LME carries poor response to immune-chemotherapy (Kotlov-Cancer-Discovery-2021), we tested anti-CD19 CAR T-cells in the models. While CAR T-cells were initially effective in the two DLBCL models, progression occurred earlier in DLBCL with DP-LME than in IN-LME, which remained in remission 100 days after cell infusion. Responses correlated with persistence of circulating CD4 + CAR T cells. These data highlight that a TP53-driven DP-LME may limit immunotherapy activity in DLBCL, providing a scientific explanation to the poor outcome of DLBCL patients with TP53 alterations to CD19-directed CAR T cells (Shouval-JCO-2021). Collectively, our study defines the cellular origins and the stepwise genetic alterations of indolent and aggressive lymphomas with MYD88 L265P, dissects distinct immunological paths underlying progression driven by oncogenic MYD88, and builds proof-of-concept for advancing precision immunotherapy in B-cell lymphomas.

A High-Risk Subgroup Multiple Myeloma Classification Based on the Detection of PR Minor Subclones

In the last two decades several classifications of Multiple Myeloma (MM) have been proposed based on gene expression signatures. These include the supervised translocation and cyclin D (TC) molecular subgroup approach or unsupervised methods to elucidate the molecular heterogeneity of MM and identify high risk subgroups. In particular, two RNA based gene expression studies have identified 8 to 10 unique subtypes, including the MS, MAF, CD1, CD2, and PR subtypes. The PR subgroup represents a highly proliferative gene signature that is associated with very poor survival outcomes. However, assigning this PR subgroup based on bulk transcriptome signatures can be challenging giving the mixture of genetic backgrounds that can be observed within this subtype. Furthermore, the presence of a minor PR subclone can often be missed with bulk genomic studies leading to the misclassification of a patient as standard risk. We here postulated that minor PR subclones expand under selective therapeutic pressures to become the predominant clones at subsequent relapses and the presence of PR subclones can impact any of the molecular subgroups pertaining poor survival outcomes. Bulk RNA or single cell RNA (scRNAseq) analysis were performed in bone marrow sorted primary MM cells obtained from 718 patients from the CoMMpass trialand 32 relapse/refractory MM patients, respectively. For scRNAseq, serial samples with paired baseline and relapse samples were available in 18 cases. Bone marrow plasma cells samples were separated from bone marrow aspirates using positive sorting selection of CD138 + cells. For bulk RNA-seq we used the mRNA HyperPrep kit (Kapa Biosystems) with RNA from 50,000 cells.For scRNA studiesunbiased mRNA profiling was conducted using the GemCode system (10x Genomics). Cell Ranger and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3' gene and data analysis. Sequencing was performed on Illumina. By performing GSEA at the single cell level using the Zhan gene data set we have classified patients in the appropriate TC groups. FISH data was used to insure accurate calls. For the survival analysis OS was analyzed by the Kaplan-Meier method. In the CoMMpass dataset, with bulk RNA sequencing, the PR subtype contained 7.1% of patients with an admixture of classic genetic subtypes and very poor clinical outcome, with a median OS of 21.3 months. High proliferation index scores were concentrated in this subtype. There was also a strong tendency for patients to transition to the PR subtype at progression, with 25.5% of patients in a non-PR subtype at diagnosis transitioning to PR. Regardless of original subtype, patients that transitioned to the PR subtype rapidly succumbed to their disease with a median OS after the detected progression of 88 days. Using subtype prediction weight we could identify non-PR patients with PR subtype weights above 5% and these patients have a significantly shorter OS of 50 months compared to patients with <5% PR. At the single cell level, investigating the relapsed patients cohort, we have analyzed 92,139 cells and identified 59 clusters. A PR signature (defined as present in ≥15% of cells) was identified in 38% of cases despite baseline favorable prognostic group by FISH cytogenetics (7% HP, 28% t(11;14), and 57% normal FISH). A good concordance with FISH data was observed. In serially relapsed paired samples, all the PR patients maintain their signature overtime and 44% of patients transitioned to PR at subsequent relapse (Fig1A). Survival analyses were performed to evaluate how the presence of minor PR clones at the single cell level relate to clinical outcomes. Notably, the overall survival of patients classified as PR was dramatically reduced with a median OS of 6 months compared to 34 months for non-PR patients (Fig1B). In conclusion, we here show that minor PR clones defined by transcriptional signature through bulk or scRNA may be detectable in patients with variable MM molecular subgroups, including those classified as standard risk by the currently adopted classifications. We also show that the presence of these PR minor clones pertains adverse outcomes with very poor survivals. At relapse, most acquired PR cases originated from standard-risk presentations consistent with a Darwinian clonal evolution. Future classifications should account for the presence of these PR subclones for a better disease prognostication and developments of novel therapeutics.