Faculty Bibliography

ARID2 is the most recurrently mutated SWI/SNF complex member in melanoma; however, its tumor-suppressive mechanisms in the context of the chromatin landscape remain to be elucidated. Here, we model ARID2 deficiency in melanoma cells, which results in defective PBAF complex assembly with a concomitant genomic redistribution of the BAF complex. Upon ARID2 depletion, a subset of PBAF and shared BAF-PBAF-occupied regions displays diminished chromatin accessibility and associated gene expression, while BAF-occupied enhancers gain chromatin accessibility and expression of genes linked to the process of invasion. As a function of altered accessibility, the genomic occupancy of melanoma-relevant transcription factors is affected and significantly correlates with the observed transcriptional changes. We further demonstrate that ARID2-deficient cells acquire the ability to colonize distal organs in multiple animal models. Taken together, our results reveal a role for ARID2 in mediating BAF and PBAF subcomplex chromatin dynamics with consequences for melanoma metastasis.

BACKGROUND:Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS:We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS:Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS:Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers^1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.

Resistance to therapy is one of the most significant challenges in the treatment of acute myeloid leukemia (AML). While great efforts have uncovered genetic mechanisms of resistance to certain AML-directed therapies, to date, treatment resistance in AML has only partially explained by acquired genetic alterations. Here, we performed genome-wide CRISPR/Cas9 screens to identify drug-gene interactions that modulate therapeutic response to treatments commonly used in AML. Interestingly, our findings uncovered several genes that regulate pre-mRNA splicing whose loss strongly synergized with venetoclax, a BH3 mimetic that blocks the antiapoptotic protein BCL-2. To further delineate the role of RNA processing in response to AML treatments, we performed secondary CRISPR screens with a domain-focused gRNA library targeting 490 RNA processing factors in the presence of various AML drugs. Overall, these genetic screens identified a number of RNA splicing factors whose loss-of-function sensitized AML cells to BCL2 inhibition (Fig. A). Among the top gene candidates whose loss promoted venetoclax efficacy was the splicing factor RBM10 (Fig.B). Strikingly, loss of RBM10 exclusively synergized with venetoclax-based treatments across AML therapeutics, including in TP53 mutant lines (Fig.C-D). Moreover, RBM10 loss restored venetoclax sensitivity to AML cell line variants with acquired venetoclax resistance. Interestingly, while many RNA splicing factors are pan-essential, generation of an Rbm10 conditional knockout mouse revealed that Rbm10 is completely dispensable for steady-state normal hematopoiesis (Fig.E). Since RBM10 has not been studied previously in hematopoiesis, we mapped the impact of RBM10 on mRNA expression and splicing using RNA-seq and direct RNA binding partners genome-wide by eCLIP-Seq (Fig. F). RBM10 loss was strongly associated with downregulation of BCL2A1, an anti-apoptotic factor whose expression is correlated with venetoclax resistance in AML (Fig.G-H). This was dependent on RBM10's ability to bind RNA and expression of BCL2A1 cDNA fully rescued the growth-inhibitory effect of RBM10 KO-venetoclax treated AML cells. Overall, the above data support RBM10 as a synthetic lethal vulnerability in venetoclax therapy. Beyond RBM10, our genetic screens also identified several splicing factors belonging to the family of serine and arginine-rich (SR) proteins whose loss synergized with venetoclax treatment (Fig. I). SR proteins are essential for pre-mRNA splicing and are substrates for phosphorylation by conserved family of kinases, such as Cdc2-like kinases (CLKs) and (dual-specificity tyrosine-regulated kinases) DYRKs. We therefore utilized a series of selective pan-CLK/DYRK1A inhibitors, including SM09419 and SM08502, that potently suppress SR protein phosphorylation. Interestingly, BCL2 is one of the top genetic dependencies upon DYRK1A genetic suppression in prior work from the DepMap (Fig. J). Pharmacologic inhibition of CLK/DYRK1A exhibited high in vitro efficacy at nanomolar range across a diverse range of AML subtypes including cell lines with acquired venetoclax resistance (Fig.K). Consistent with this, combined SM09419 and venetoclax displayed synergistic anti-leukemic effects and venetoclax-sensitive AML cell lines (Fig.L). Taken together, these data support the notion of targeting CLK/DYRK1A in the context of BCL2 inhibition. In this study, we systematically defined gene interactions that mediate the response to a wide range of AML drugs. Recent studies have begun to show that dysfunctional RNA processing promotes AML development. However, the role of RNA processing in modulating drug responsiveness in AML is not well understood. Here, we have uncovered that synthetic lethal targeting of splicing factors, such as RBM10, increases sensitivity of AML cells to BCL2 inhibition. Therapeutically, pharmacologic inhibition of SR protein function via inhibiting CLK/DYRK1A-mediated phosphorylation of splicing factors is an effective strategy used in combination with venetoclax or to overcome venetoclax resistance. Overall, our findings underscore the central importance of RNA splicing in drug response and provides a therapeutic rationale for modulating RNA splicing to enhance current AML therapies. [Formula presented] Disclosures: McMillan: Prizer: Ended employment in the past 24 months. Bossard: Biosplice Therapeutics: Current Employment. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.
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Background Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer and while curable in the majority of cases, 15%-20% of children will relapse with only 50% surviving long-term. Treatment failures arise from the outgrowth of pre-existing or acquired sub-clones that are genetically or epigenetically primed to resist treatment. In addition, the bone marrow microenvironment is known to provide a protective niche. We performed the first mapping of the human B-cell ALL (B-ALL) immune bone marrow (BM) microenvironment at single cell resolution at diagnosis, remission and relapse (Witkowski, 2020). We uncovered a striking rewiring of the myeloid compartment during B-ALL progression with significant over-representation of a leukemia-associated monocyte subpopulation expressing high levels of the Macrophage Colony Stimulating Factor Receptor (M-CSFR/CSF1R). Using both peripheral blood (PB) complete blood count analysis and RNA-seq data, we demonstrated that high monocyte abundance at B-ALL diagnosis is predictive of inferior pediatric and adult overall survival in two large independent clinical cohorts. To determine the association of non-classical monocyte abundance in BM and PB with risk of relapse, we examined a cohort of clinical samples from children enrolled on Children's Oncology Group (COG) protocols. Methods Using an unmatched case-control design, we established a preliminary cohort of PB and BM samples collected at diagnosis from 24 B-ALL patients with eventual relapse and 24 patients in long-term remission. Four remission samples from an NYU Langone cohort were used to validate the expansion of this population in the presence of B-ALL. We applied a customized flow cytometry based assay to identify CD115-expressing human monocyte subsets: classical (CD45 ⁺CD56 ^-CD14 ⁺CD16 ^-), non-classical (CD45 ⁺CD56 ^-CD14 ^-CD16 ⁺), as well as B-cells (CD19, CD22, CD10) and T/NK cells (CD3, CD56). We then performed univariate and multivariable analysis of outcome (relapse versus long-term remission) compared to monocyte subset abundance, adjusting for potential confounding factors (age, gender, CNS status, NCI risk, genetic subtype, WBC at diagnosis, and end of induction minimal residual disease). Results We observed a significantly higher percentage of non-classical monocytes in the diagnostic BM from the COG cohort when compared to remission samples (COG diagnostic B-ALL BM non-classical percentage mean 52.19% vs NYU B-ALL remission BM non-classical percentage mean 1.775%, P = 0.0001). We also observed a strong correlation between the percentage of non-classical monocytes in the PB when compared to their matched BM specimens (r = 0.6, P = 0.0001). Multivariable analysis revealed a significant association between PB non-classical monocyte percentage at diagnosis and patient outcome (remission cohort non-classical monocyte percentage [mean, range]: 52.4%, 33.3-68.1%, n = 24, relapse cohort non-classical monocyte percentage: 65.9%, 56.4-84.7%, n = 24, P = 0.021). Similarly, a strong trend was observed in the BM, although it did not reach statistical significance. Flow cytometric analysis confirmed CD115 (M-CSFR/CSF1R) expression in this non-classical monocyte population, thereby validating a novel target for intervention. Conclusions These findings validate the presence of a unique monocyte subpopulation associated with childhood B-ALL and suggests that assessing this population in PB at diagnosis may be of prognostic significance. The availability of small molecule inhibitors and monoclonal antibodies targeting CSF1R-expressing monocytes may offer a novel approach to treating B-ALL. [Formula presented] Disclosures: Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: Sobi: Consultancy; NeoImmune Tech: Research Funding; Janssen: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy.
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Multiple myeloma (MM) is a plasma cell malignancy characterized by the presence of multiple foci in the skeleton. These distinct tumor foci represent cycles of tumor growth and dissemination that seed new clusters and drive disease progression. By using an intratibial Vk*MYC murine myeloma model, we found that CD169+ radiation-resistant tissue-resident macrophages (MPs) were critical for early dissemination of myeloma and disease progression. Depletion of these MPs had no effect on tumor proliferation, but it did reduce egress of myeloma from bone marrow (BM) and its spread to other bones. Depletion of MPs as a single therapy and in combination with BM transplantation improved overall survival. Dissemination of myeloma was correlated with an increased inflammatory signature in BM MPs. It was also correlated with the production of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by tumor-associated MPs. Exogenous intravenous IL-6 and TNFα can trigger myeloma intravasation in the BM by increasing vascular permeability in the BM and by enhancing the motility of myeloma cells by reducing the adhesion of CD138. Moreover, mice that lacked IL-6 had defects in disseminating myeloma similar to those in MP-depleted recipients. Mice that were deficient in TNFα or TNFα receptor (TNFR) had defects in disseminating MM, and engraftment was also impaired. These effects on dissemination of myeloma required production of cytokines in the radiation-resistant compartment that contained these radiation-resistant BM MPs. Taken together, we propose that egress of myeloma cells from BM is regulated by localized inflammation in foci, driven in part by CD169+ MPs.

T cells, the main effectors of the adaptive immune system, originate from thymocyte precursors in the human thymus. These immature cells follow a tediously regulated differentiation process with several critical checkpoints to ensure the generation of a diverse repertoire of fully functional, non-self T cells. In previous studies, we already uncovered part of the regulatory mechanisms that drives this differentiation process in human, which is notably different compared to mouse (De Decker et al., 2020; Dolens et al., 2020; Lavaert et al., 2020; Roels et al., 2020a; Roels et al., 2020b). However, a lot of the regulatory complexities remain unknown. By profiling additional layers of these discrete stages of thymocytes, we here aimed to generate the most complete integrative multi-omics reference map of human T cell development so far. By using a multi-omics factor analysis approach, we combined transcriptomics, epigenomics, proteomics and finally 3D genomics into one model to fully describe the T cell developmental process. We found that human T cell development is equally affected by previously unappreciated drivers, including non-coding and circular RNA, alternative splicing and DNA methylation. While some omics layers show global and genome-wide changes within developing alphabeta and gammadelta T cells, the global 3D structure remains very stable throughout all differentiation states. Nonetheless, specific alterations do occur at developmentally important genes, especially at the boundaries of active and inactive compartments. This integrative analysis aids our understanding of immune cell development both in health and disease, and reveals important aspect of lineage differentiation dynamics

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.