Search

Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Molecular glue compounds induce protein–protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines, we identify CR8—a cyclin-dependent kinase (CDK) inhibitor—as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12–cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous “living drugs,” these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4 CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify “super-degron” tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

While a number of targeted therapies have recently become available in the treatment of acute myeloid leukemia (AML), it remains to be determined whether early acquired mutations are essential for ongoing leukemia maintenance in the face of sequential acquisition of additional mutations. In some cases, our ability to answer this question is hampered by the lack of targeted therapies to a number of these mutations. Of all AML mutations, those involving DNMT3a and NPM1c are some of the most frequent - often acquired before the final transforming event. Here we report the development of an in-vivo degron-based approach to interrogate the requirement for DNMT3a-R882 and NPM1c after leukemia development. Using an IKZF-based degron system offers us the opportunity to address the ongoing requirement of particular oncogenes for AML maintenance - by way of a conditional system that can be rapidly modulated. Thalidomide and its related analogues act by recruiting various substrates to the CRL4CRBN E3-ubiquitin ligase complex mediating subsequent ubiquitination and proteasomal degradation. In a majority of cases - the basis of this degradation has been determined to be the result of recognition and binding to a degron motif within C2H2 zinc fingers. Using more detailed analysis with single amino-acid resolution, we have engineered 'degrons' sensitive or resistant to degradation by thalidomide and its related analogues. In-vivo degradation using pomalidomide revealed a dependence of GMP cells on the presence of NPM1c but not DNMT3a R882. Using our cereblon (CRBN) knock-in mice engineered to mediate sensitivity to these compounds, we generated triple-mutant acute-myeloid leukemia - sequentially introducing DNMT3a-R882, NPM1c and N-RAS G12D mutations whilst altering the site of the degron tag in each case. Degradation-resistant degron constructs were used as a control in each case. Findings were subsequently correlated and validated with in-vitro treatments of triple mutant cells. RNA-seq was also performed after both in-vitro and in-vivo treatments to further delineate acute transcriptional changes in the absence of NPM1c - highlighting a number of changes including those within the Hox family of genes Together, these findings suggest triple mutant leukemia remains dependent on the leukemia-inducing mutation of NPM1c. In contrast, whilst epigenetic alterations induced by DNMT3a mutations are essential for leukemia development - these do not appear to be essential for ongoing leukemia maintenance. Further work is currently underway to determine how modulating NPM1c impacts long-term survival from leukemia and whether the epigenetic changes associated with DNMT3a R882 mutations are indeed reversible in the context of leukemia. This work highlights an approach to interrogate a protein of interest in-vivo using a conditional system that is reversible, rapidly acting and that uses a series of FDA-approved compounds with known kinetics. We envisage this will be generalisable as a tool to be used in other similar contexts. Disclosures No relevant conflicts of interest to declare.

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

The discovery that thalidomide derivatives recruit the E3 ligase CRBN to induce neomorphic degradation of proteins critical for multiple myeloma (MM) cells stimulated the research into proteolysis-targeting chimeric compounds (PROTACs), led to development of several CRBN- or VHL-based PROTACs against various oncoproteins and put a new spotlight on the biology and therapeutic targeting of E3 ligases in human neoplasias. However, so far only a few of the ~600 known/presumed E3 ligases have been leveraged for generation of PROTACs. The mechanisms regulating the function of most E3 ligases have not been systematically examined. Because the function of an E3 ligase is considered essential for anti-tumor activity of its respective PROTACs, we applied CRISPR knock-out (KO) systems to identify candidate regulators of E3 ligase function, via characterizing the the network of genes which modulate MM cell responses to PROTACs. We thus performed genome-scale CRISPR-based gene editing (for loss-of-function, LOF) studies in MM.1S cells treated with PROTACs targeting BET bromodomain proteins through MDM2 (A1874), CRBN (dBET6) or VHL (ARV-771 or MZ-1) or targeting CDK9 through CRBN (Thal-SNS-032); and validated key hits with individual sgRNAs in different MM cell lines. The top individual LOF events conferring resistance to PROTACs did not involve a compensatory mechanism or "work-around" the loss of the respective oncoprotein, but were predominantly associated with LOF of the respective E3 ligase; or with LOF for genes with known or plausible role in regulating the respective E3 ligases. For instance, sgRNAs against members of the COP9 signalosome complex decreased MM cell responses to CRBN- and (to a lesser extent) VHL-, but not MDM2-based PROTACs. PROTACs leveraging different E3 ligases were regulated by different cullin ring ligase (CRL) complex members (e.g. CUL2, RBX1, TCEB1, TCEB2 for VHL- vs. DDB1 for CRBN- vs. no CRL member for MDM2-based PROTACs) or E2 conjugating enzymes (UBE2R2 vs. UBE2G1 for VHL- vs. CRBN-based PROTACs). Collectively, these results suggest that MDM2 regulation is largely CRL- and COP9-signalosome independent; while VHL regulation is less COP9 signalosome-dependent compared to CRBN. These mechanistic differences suggest that PROTACs targeting the same oncoprotein through different E3 ligases should not be associated with cross-resistance, a result which we validated in experiments involving sequential administration of different PROTACs against BRD4/3/2. In turn, this observation implied that developing PROTACs that leverage a more extended spectrum of E3 ligases may facilitate sequential uses of existing and these new PROTACs to delay or prevent treatment resistance. Building on results of our genome-scale CRISPR essentiality screens, we examined the dependency landscape of known E3 ligases of MM (n=20 cell lines) and 500+ non-MM cell lines. CRBN is redundant for nearly all MM or non-MM cell lines tested, while most other E3 ligases leveraged for PROTACs (e.g. MDM2, BIRC2, DCAF15, DCAF16, RNF114) are essential for only modest or small subsets of human cancer cell lines, suggesting that resistance to respective PROTACs may readily emerge through LOF of these E3 ligases without major fitness cost to tumor cells. We thus sought to identify E3 ligases which are highly expressed in subsets of human tumor cell lines (at levels well above the large majority of normal tissues) and are major dependencies for these "high expressor" cell lines: we identified MDM2 as a major dependency for p53-wild-type cell lines (consistent with MDM2 role as E3 ligase for p53) and we validated this result by documenting the preferential activity of a MDM2-based PROTAC for BRD4/3/2 against p53 wild-type cells. We also identified other E3 ligases genes with well-known roles in tumor cell biology (e.g. members of anaphase promoting complex/cyclosome); as well as E3 ligases (e.g. KCMF1, RNF4) which, to our knowledge, have not been leveraged for design of PROTACs, but warrant consideration given their patterns of essentiality in "high expressor" tumor cells. Our study provides insights on differential regulation and distinct patterns of essentiality for different E3 ligases and informs the design of new PROTACs which leverage different E3 ligases to help delay/overcome treatment resistance in MM and beyond. Disclosures Schlossman: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Celgene: Research Funding; Deerfield: Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological “degraders” of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

The small molecule drugs thalidomide, lenalidomide, and pomalidomide induce ubiquitination and proteasomal degradation of the zinc finger transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruitment to Cereblon (CRBN), the substrate receptor of the CRL4CRBNE3 ubiquitin ligase.Here we screened the entire human Cys2-His2 (C2H2) zinc finger proteome for degradation by CRL4CRBN in the presence of thalidomide analogues, identifying 11 zinc finger targets. Structural and functional characterization of the C2H2 zinc finger degron demonstrates how diverse zinc finger domains bind the promiscuous drug-CRBN interface. Computational ZF docking, in conjunction with biochemical analysis, predicts that at least 50 zinc-fingers bind the drug-CRBN complex in vitro, a larger number than previously anticipated. Functional studies demonstrate that selective degradation of zinc fingers could be tuned through compound modifications, providing a basis for the development of small molecules that degrade a wide range of zinc finger transcription factors.

The small molecule drugs thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of Ikaros (IKZF1) and Aiolos (IKZF3) by mediating their interaction with Cereblon (CRBN), the substrate receptor of the CRL4CRBN ubiquitin ligase. Here we screened the human Cys2-His2 (C2H2) zinc finger (ZF) proteome for degradation by CRL4CRBN in the presence of thalidomide analogues, identifying 11 ZF targets. Structural and functional characterization of the C2H2 zinc finger degron demonstrates how diverse ZF domains bind the drug-CRBN interface. Computational ZF docking, in conjunction with biochemical analysis, predicts that at least 50 zinc-fingers bind the drug-CRBN complex in vitro, a larger number than previously anticipated. These results provide strategies to degrade other zinc finger transcription factors. Citation Format: Quinlan L. Sievers, Georg Petzold, Richard D. Bunker, Aline Renneville, Brian Liddicoat, Wassim Abdulrahman, Tarjei Mikkelsen, Benjamin L. Ebert, Nicolas H. Thoma. The zinc-finger degrome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL02-03. doi:10.1158/1535-7163.TARG-19-PL02-03

Thalidomide-targeted degradation Thalidomide and its analogs improve the survival of patients with multiple myeloma and other blood cancers. Previous work showed that the drugs bind to the E3 ubiquitin ligase Cereblon, which then targets for degradation two specific zinc finger (ZF) transcription factors with a role in cancer development. Sievers et al. found that more ZF proteins than anticipated are destabilized by thalidomide analogs. A proof-of-concept experiment revealed that chemical modifications of thalidomide can lead to selective degradation of specific ZF proteins. The detailed information provided by structural, biochemical, and computational analyses could guide the development of drugs that target ZF transcription factors implicated in human disease. Science , this issue p. eaat0572

Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4

Cell-based therapies are emerging as potent agents against cancer and other diseases, but are uniquely uncontrolled "living drugs". For example, chimeric antigen receptor (CAR) T cells can effectively target hematologic malignancies yet pose a risk for toxic hyperactivation. Future cell-based therapies, including CAR T cells, could be improved by incorporating specific and reversible control systems. However, clinically suitable ON- and OFF-switches engineered from non-immunogenic human polypeptide sequences and regulated by non-immunosuppressive FDA-approved drugs are needed. Here we report the engineering of a robust lenalidomide-responsive degron tag, which we then used to construct a degradable CAR affording reversible OFF-switch functional control at clinically relevant lenalidomide doses. Thalidomide, lenalidomide, and pomalidomide are effective and clinically approved therapies for multiple myeloma, subtypes of non-Hodgkin lymphoma, and myelodysplastic syndrome with chromosome 5q deletion. These drugs exert therapeutic properties by acting as molecular glue, bridging interactions between the CRL4CRBN ubiquitin ligase and disease-relevant proteins that are subsequently ubiquitinated and degraded by the proteasome. A set of Cys2-His2 (C2H2) zinc fingers have emerged as degron motifs mediating drug-dependent interactions with the CRL4CRBN ubiquitin ligase. We hypothesized that these small, modular, human polypeptide domains could be further engineered and repurposed as tags to induce drug-dependent depletion of engineered proteins. By systematically shuffling the subdomains of all known zinc finger degrons and functionally screening this hybrid zinc finger library, we engineered "super-degron" tags that are more efficiently degraded at lower drug concentrations than any C2H2 zinc finger in the human proteome. We then incorporated one of these super-degrons (SD01) into a second-generation CAR targeting CD19 (FMC63-4-1BB-CD3z), thereby constructing a degradable CAR (Figure 1A). In a Jurkat T cell model, addition of lenalidomide induced rapid and near-complete depletion of the degradable CAR (Figure 1B/C). When co-cultured with target cells expressing CD19, therapeutically relevant lenalidomide concentrations robustly inhibited T cell activation (Figure 1D). Indeed, the IC50 for inhibition of IL2 secretion and CD69 expression were 2 and 22 nM lenalidomide, respectively, whereas in myeloma patients the plasma concentration of lenalidomide following one 25 mg oral dose decays from ~1000 to 10 nM over the course of 24 hours (Connarn et al, CPDD, 2017). In primary T cells, 100 nM lenalidomide suppressed degradable CAR effector functions including tumor cell killing and cytokine release (Figure 1E). Using pomalidomide, which has a longer in vivo half-life, we demonstrated robust and reversible depletion of the degradable CAR in T cells engrafted in NSG mice. Together, these findings demonstrate reversible OFF-switch control of degradable CARs at clinically relevant lenalidomide concentrations. Experiments are underway to determine whether, in the context of tumor clearance in NSG mice, degradable CAR T cell effector functions can be paused and subsequently released with short-term administration of lenalidomide. Whereas the current management of CAR T cell hyperactivation syndromes consists of supportive care, tocilizumab, and/or high-dose corticosteroids, we propose that cytokine release and CAR-related encephalopathy syndromes may be more easily diagnosed and managed with degradable CARs. The super-degron tags presented here are generalizable and clinically suitable tools to achieve chemical genetic control of diverse genetically engineered cell therapies. Disclosures Jan: Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Sievers:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Maus:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Deerfield: Research Funding; Celgene: Research Funding.

Background Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. Methods We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. Results At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. Conclusion Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. Trial registration Clinicaltrials.gov NCT00442130. Funding NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Summary Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Multiple Myeloma (MM) remains incurable in part due to an incomplete understanding of the genes critically responsible for MM cell survival and proliferation. We reasoned that CRISPR/Cas9-based functional genomics could identify genes essential for survival or proliferation of MM cells and define candidate therapeutic targets. We transduced Cas9-expressing RPMI-8226 and MM.1S cells with lentiviral genome-scale GeCKO pooled libraries of sgRNAs. After culture of these cell lines for 2-12 weeks without treatment we identified, based on next generation sequencing, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR90% of MM patients, the candidate MM-preferential essential genes are not mutated. Some of these genes are overexpressed in MM vs. non-MM cell lines (CCLE dataset) or patient-derived samples (Broad/MMRF vs. TCGA datasets); induced by BMSC co-culture; proximal to superenhancers; or correlating with clinical outcome. However, these molecular results were not sufficient, collectively or individually, to identify this cohort of MM-preferential essential genes independently of the CRISPR functional data. Targeting lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our functional genomic studies in molecularly annotated MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Citation Format: Geoffrey M. Matthews, Ricardo de Matos Simoes, Yiguo Hu, Michal Sheffer, Olga Dashevsky, Eugen Dhimolea, Paul J. Hengeveld, Brian J. Glassner, Sara Gandolfi, Megan A. Bariteau, Quinlan Sievers, Benjamin L. Ebert, Franciska Vazquez, Aedin Culhane, Constantine S. Mitsiades. Characterization of lineage vs. context-dependent essential genes in multiple myeloma using CRISPR-Cas9 genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-118. doi:10.1158/1538-7445.AM2017-LB-118

One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.

Lenalidomide exerts its therapeutic effects in the malignancy multiple myeloma by facilitating the degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the CRL4-CRBN E3 ubiquitin ligase. In the following study we utilized a positive selection, genome-scale CRISPR-Cas9 screen in the lenalidomide-sensitive myeloma cell line, MM1S, to further our understanding of the molecular machinery which regulates and is required for lenalidomide-mediated modulation of CRL4-CRBN. The gRNAs demonstrating the greatest enrichment following selection with lenalidomide belong to cereblon, the molecular target of lenalidomide and substrate receptor for the CRL4-CRBN ubiquitin ligase. Additionally, 20 of the top 30 genes highlighted by the screen are implicated in both the positive and negative regulation of cullin-ring ligases (CRLs), emphasizing both the importance of cereblon's E3 ligase function in mediating cell death as well as the equilibrium of forces required for proper function of CRLs. Among these genes were the ubiquitin-donor E2 enzymes UBE2D3 and UBE2G1; despite the relevance of E2s to E3 ligase biology it has been difficult to determine via traditional methods which of the ~35 E2 enzymes are utilized by a given E3 ligase. Here we demonstrate that G1 and D3 fulfill distinct functional roles and cooperate with the CRL4CRBN E3 ligase to ubiquitinate an aiolos reprorter with lysine-48 linkages. In aggregate these findings confirm the power of CRISPR-Cas9 positive selection screening to reveal drug mechanism-of-action and provides a paradigm for the identification and functional characterization of E2-E3 pairings. Disclosures Fischer: Novartis: Consultancy.

Multiple Myeloma (MM) remains an incurable malignancy in part because of an incomplete understanding of which genes are critically responsible for MM cell survival and proliferation. To address this unmet need, and building on our recent functional genomics studies with the CRISPR/Cas9 gene editing platform (ASH 2015; Int. MM Workshop, Rome 2015), we reasoned that quantification of sgRNA depletion in the absence of any treatment could identify genes essential for the survival or proliferation of MM cells and better define their role as candidate therapeutic targets. To this end, we transduced Cas9-expressing RPMI-8226 and MM.1S cells with the lentiviral genome-scale GeCKO pooled library of sgRNAs. After culture of these cell lines for 2, 6, 8 or 12 weeks without any treatment, we identified, based on next generation sequencing for the sgRNA sequences, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR=0.05, based on MAGECK algorithm) in Cas9+ cells compared to their initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 50 cell lines from other hematologic malignancies (leukemia, lymphoma) and from 8 different types of solid tumors. We identified 3 broad categories of essential genes in MM cells: a) core essential genes, with sgRNA depletion across the majority of MM and non-MM lines of our study, representing cellular processes critical for practically all lineages (e.g. genes involved in regulation of basic transcription factor complexes, ribosomal function, proteasome, spliceosome, structural proteins for mitochondria and other key organelles, et.c.); b) genes selectively essential for MM cell lines, but not for the overwhelming majority of leukemia, lymphoma or solid tumor cell lines; c) genes with a role in small subset(s) of cell lines, across diseases, which harbor defined genetic features correlating with this dependency. We integrated our CRISPR/Cas9-based data on MM-selective essential genes with a reanalysis of the Achilles Heel shRNA screen in MM and non-MM cell lines (10 and 493, respectively) of the Cell Line Encyclopedia Program (CCLE) program. We applied a series of statistical tests (e.g. Wilcoxon rank test or marker selection feature of GENE-E algorithm with 1000 permutation tests) to identify genes with a significantly lower rank in sgRNA or shRNA depletion in MM vs. non-MM cell lines, across different specific thresholds for fold change and statistical significance. We identified more than 50 high-value candidate target genes with preferential essentiality in MM, compared to non-MM cell lines of diverse lineages. Prominent examples of such MM-selective, essential genes included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); otherNF-kB-related genes (e.g. IKBKB); PIM2 (but not PIM1 or PIM3 in this cell line panel); regulators of protein homeostasis, including diverse E2 and E3 ubiquitin ligases; and several other known or biologically-plausible dependencies which are under further evaluation. Many of these MM-selective dependencies exhibited significantly higher expression in MM, compared to non-MM cells, both in cell lines (based on the CCLE dataset) and patient-derived samples (comparison of Broad/MMRF vs. TCGA datasets, respectively). Notable observations of context-dependent essential genes include ARID1A in MM.1S cells (plausibly due to deficiency in its paralog ARID1B); and cases of both MM and non-MM cells with RAS mutations but lack of dependency on that gene. Targeting of lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our integrated genome-wide CRISPR/Cas9 and shRNA analyses in molecularly annotated panels of MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Disclosures No relevant conflicts of interest to declare.

Abstract 463 The somatic genetic basis of chronic lymphocytic leukemia (CLL), a common and clinically heterogenous adult leukemia, remains poorly understood. Massively parallel sequencing technology provides a method for systematic discovery of genetic alterations that underlie disease, and for uncovering new therapeutic targets and biomarkers. We therefore performed DNA sequencing of 91 CLL samples with matched germline, consisting of 88 exomes and 3 genomes. Samples were collected from patients displaying a range of characteristics representing the broad clinical spectrum of CLL. Libraries were constructed and sequenced on an Illumina GA-II sequencer to deep coverage. Point mutations and indels were detected by comparing sequences of each tumor to its corresponding normal. We detected 1828 non-silent mutations in protein-coding sequences, corresponding to an average somatic mutation rate of 0.72/Mb (range 0.075–2.14). Nine cancer genes were identified, as they were mutated significantly more often than the background rate given their sequence composition across all 91 CLL/normal pairs. Four have been previously described in CLL (TP53, ATM, MYD88 and NOTCH1); the others were novel (SF3B1, ZMYM3, MAPK1, FBXW7 and DDX3X). Strikingly, SF3B1, which functions at the catalytic core of the spliceosome, was mutated the second most frequently (15%). All 14 identified mutations localized to a discrete region (exons 12–18), and 7 were recurrent (K700E). We additionally validated the SF3B1-K700E mutation through genotyping of 101 independent CLL samples. The 9 significantly mutated genes fell into 5 core signaling pathways, in which the genes play established roles: DNA damage repair and cell-cycle control (TP53, ATM), Notch signaling (FBXW7, NOTCH1), inflammatory pathways (MYD88, DDX3X, MAPK1) and RNA splicing/processing (SF3B1, DDX3X). The common cytogenetic aberrations in CLL carry strong prognostic significance, implying that they reflect distinct pathogenesis. Supporting this hypothesis, we discovered strong associations between different driver mutations and key FISH abnormalities. Most TP53 mutations were in samples with del(17p) (p To define the impact of the mutated cancer genes on disease outcome, we constructed a Cox multivariable regression model for factors contributing to earlier time to first therapy (TTFT) in the 91 CLLs. SF3B1 mutation was predictive of poor prognosis (HR 3.17, p=0.004), independent of other established markers (i.e. IGVH mutation; del(17p); ATM mutation). Consistent with this analysis, patients harboring the SF3B1 mutation alone had short TTFT, as did patients with del(11q) or del(11q)+SF3B1 mutation. All 3 groups demonstrated significantly shorter TTFT than patients without either SF3B1 mutation or del(11q). Because SF3B1 encodes a splicing factor, we looked for functional evidence of splicing alterations associated with SF3B1 mutation. Indeed, mRNA targets of the spliceosome exhibited greater intron retention in CLLs with mutated vs nonmutated SF3B1, confirming alteration in normal SF3B1 function. Understanding the mutational landscape of CLL provides a starting point for systematic analyses to address fundamental questions in CLL, including how mutated genes alter cellular networks and phenotypes, and thereby contribute to disease heterogeneity. Our discovery of striking associations between driver mutations and standard CLL prognostic markers (cytogenetic aberrations, IGVH mutational status) suggest synergistic interactions. In particular, our studies highlight pre-mRNA splicing as a critical but thus far unexplored cellular process contributing to CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 693 The molecular pathogenesis of chronic lymphocytic leukemia (CLL) has not been well-characterized. Next generation sequencing technologies provide an unprecedented opportunity to screen entire genomes for genetic changes associated with tumorigenesis. To examine the mutational spectrum in CLL, we completed Illumina sequencing of 2 genomes and 4 exomes from patients with advanced CLL in parallel with germline DNA from autologous skin fibroblasts (average coverage 66x/59x for CLL/normal). While the somatic mutation rates varied widely between individuals, ranging from 1–10 per megabase, we discovered that 2 of 6 tumors harbored novel nonsynonymous mutations in Wnt pathway members (DKK2 [R197H] from Patient 1 and BCL9 [G458S] from Patient 3), suggesting that perturbation of this pathway may be critical in CLL. By targeted quantitative RNA sequencing, we detected both mutated DKK2 and BCL9 as heterozygous mutations in tumor RNA from Patients 1 and 3, respectively. We confirmed that the Wnt pathway is in fact highly dysregulated in CLL based on gene expression analysis of 141 Wnt pathway members, in a dataset comprised of 155 CLL and 20 normal CD19+ B cell samples. In CLL cells, Wnt family members (Wnt1, Wnt2, Wnt2B, Wnt3, Wnt4, Wnt5B, Wnt9A, Wnt10A, and Wnt16) and Wnt receptors (FZD3, FZD8, FZD9, LRP6) were overexpressed. Additionally, we observed downregulation of Wnt pathway repressors (TLE1, SOX4 and NKD1) and upregulation of activators (SMAD2, DVL2, DVL3 and PYGO1). Expression of the downstream transcription factor LEF1 was strikingly elevated in CLL, and we measured consistent 3–4 fold elevation of LEF1 protein in CLL-B cells (n=10) compared to normal B cells (n=5) by intracellular staining (p= 0.002). Patient CLL cells demonstrated 5-fold greater beta-catenin mediated activation of the Wnt pathway in CLL compared to normal B cells (p=0.01), as measured by direct introduction of the LEF1/TCF1 luciferase reporter genes into primary cells. Silencing of LEF1 in CLL cells (n=5) led to increased cell death in vitro by 70–90%. Collectively, these results demonstrate that Wnt pathway activation and dysregulation contributes to CLL survival. To examine the possible effects of mutated DKK2 and BCL9 on Wnt pathway activity, we introduced plasmids encoding wild-type or mutated genes, together with those encoding Wnt1 protein and Wnt reporter genes, into 293T cells. DKK2 is a secreted factor that normally represses Wnt activation by competing with Wnt proteins for binding to its surface receptors. We observed that DKK2 [R197H] is mutated in its second cysteine-rich domain, which is necessary for its interaction with the Wnt receptor. BCL9 interacts with the LEF1/TCF transcriptional factor complex and has been described to have both repressive and activating effects. In 3 of 3 experiments, we observed that the mutated forms of both genes lost their repressive activity. The loss of repression generated for both mutated genes was most marked in the presence of low concentrations of Wnt protein. Moreover, both mutated genes act as dominant alleles, especially in the presence of low concentrations of Wnt protein. Since DKK2 is a secreted factor, we examined the effects of plasma of Patient 1 on Wnt pathway activity. In both normal donors (n=4) and CLL patients (n=4), DKK2 protein could be consistently detected at equivalent amounts in blood plasma following immunoprecipitation and immunoblotting with DKK2-specific antibodies Co-culture of 293T cells expressing the Wnt pathway luciferase reporters with Patient 1 plasma (2-10%) resulted in dramatic elevation of Wnt activation, which was abolished by prior depletion of DKK2. We observed similar Wnt activation when testing plasma from 22 of 93 CLL patients, but not from 5 normal volunteers. Further sequencing of 100 Wnt pathway members from 60 additional CLL patients is in progress to define the extent to which somatic mutation is a common mechanism by which this pathway is altered. Our studies suggest that Wnt pathway inhibitors represent a promising therapeutic approach for CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2417 Marrow is a major site of disease development and progression for chronic lymphocytic leukemia (CLL), as well as a priming site for antigen-specific T cells and a reservoir for memory T cells. To determine the extent to which T cells in the marrow microenvironment have an altered phenotype and function in CLL, we analyzed the immunophenotypic characteristics of marrow-infiltrating T cells of 18 CLL patients compared to 11 normal donors. Chemotherapy-naïve CLL patients (n=7) possessed comparable quantities of marrow T cells compared to normal donors (median CD8+ T cells/μl = CLL 904 vs normal 1247; median CD4+ T cells/μl = CLL 1975 vs normal 1110). However, we identified several aberrant characteristics among T cells infiltrating the marrow of CLL patients. First, the ratio of CD8+ to regulatory T cells (CD4+CD25+FOXP3+) was depressed (median ratio CLL 14 vs normal 41), indicating more regulatory T cells per effector T cells in CLL. Second, compared to normal marrow T cells, CLL marrow contained proportionally fewer functional effector CD8+ T cells (CD27+CD28+)(median normal 57%, CLL 48%) and more immunosenescent cells (CD27-CD28-)(median normal 21%, CLL 30%). Third, the T cell differentiation state of CLL CD8+ T cells was skewed to favor a phenotype of increased terminal differentiation (CD45RA+CCR7-)(median CLL 55% vs normal 40%), and decreased naïve (CD45RA+CCR7+) cells (median CLL 21% vs normal 31%) compared to normal donors. These differences were further accentuated in CLL samples collected within 4 months from treatment with conventional chemotherapy (n=11). Finally, by immunohistochemical staining of CLL marrow biopsies, we observed marrow-infiltrating lymphocytes to express PD-1 (mean of infiltrating T cells, untreated CLL 12%, treated CLL 35%, present even >6 months after therapy), a marker associated both with immuno-activation and inhibition. While the majority of PD-1+ CD8 T cells of normal donors (n=5) and treated CLL patients (n=4) were differentiated towards effector memory (CD45RA-CCR7-) cells (median normal 46% vs untreated CLL 16%, p=0.07; treated CLL 61%), the PD-1+ T cells from untreated CLL patients (n=5) were terminally differentiated (CD45RA+CCR7+)(median normal 23% vs untreated CLL 65%, p=0.04; treated CLL 24%). These results indicate an exhausted rather than an activated T cell phenotype in untreated patients. Paired immunophenotypic analysis on blood and marrow from the same individuals (n=9) demonstrated an increased percentage and intensity of PD-1 expression on T cells from marrow compared to blood (percentage CD8+ T cells BM vs blood p = 0.05). Interestingly, PD-1 was also detected on CLL cells (n=16) but not normal B cells (median normal 0%, vs CLL 17%, p = 0.004). The ligand for PD-1, PD-L1, was detected in the marrow vasculature by immunohistochemical staining of biopsies, suggesting that the marrow microenvironment plays a role in the induction of PD-1 associated immunosuppression. Ligation of blood PD-L1 on CLL-T cells led to a 2-fold decrease in activation (measured as CD69 expression) of CD3/CD28 stimulated patient T cells. In summary, we identify several phenotypic and functional alterations within marrow-infiltrating T cells of CLL patients. We speculate these together may contribute to impaired priming of host immunity against the tumor. The PD-1 pathway appears to be activated in CLL, especially in the setting of chemotherapeutic treatment. Since anti-PD1 antibodies are now clinically available, it may be possible to target this pathway to improve anti-tumor responses. Disclosures: No relevant conflicts of interest to declare.