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
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.
Thalidomide analogs exert their therapeutic effects by binding to the CRL4
E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. ...Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with
and
in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both
and
. Our findings suggest that patients with hematologic malignancies harboring these
fusion proteins may benefit from avadomide treatment.
•Targeted RNA sequencing detected a cryptic G3BP1-PDGFRB rearrangement in a myeloid neoplasm with eosinophilia and normal FISH studies.•Consistent with the patient's response to imatinib, we ...demonstrate this rearrangement is oncogenic and sensitive to TKI in cell culture.
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.
Display omitted
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.
Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue ...origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.
The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific ...epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.
Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific ...methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.
PDF
