PRMT5 supports multiple oncogenic pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with an overall poor prognosis, particularly for patients that progress on targeted therapies. Novel, more durable treatment options are needed for patients with MCL. Protein arginine methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and posttranslational modification of cell cycle regulators, DNA repair genes, components of prosurvival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here, we report on the antitumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in xenografts derived from patients with MCL. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of B-cell receptor-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and wild-type TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.

PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

LP-118 is a novel B-cell lymphoma 2 / extra-large inhibitor that demonstrates efficacy in models of venetoclax-resistant chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) respond well to initial treatment with the Bcell lymphoma 2 (BCL2) inhibitor venetoclax. Upon relapse, they often retain sensitivity to BCL2 targeting, but durability of response remains a concern. We hypothesize that targeting both BCL2 and B-cell lymphoma-extra large (BCLXL) will be a successful strategy to treat CLL, including for patients who relapse on venetoclax. To test this hypothesis, we conducted a pre-clinical investigation of LP-118, a highly potent inhibitor of BCL2 with moderate BCLXL inhibition to minimize platelet toxicity. This study demonstrated that LP-118 induces efficient BAK activation, cytochrome C release, and apoptosis in both venetoclax naïve and resistant CLL cells. Significantly, LP-118 is effective in cell lines expressing the BCL2 G101V mutation and in cells expressing BCLXL but lacking BCL2 dependence. Using an immunocompetent mouse model, Eµ-TCL1, LP-118 demonstrates low platelet toxicity, which hampered earlier BCLXL inhibitors. Finally, LP-118 in the RS4;11 and OSU-CLL xenograft models results in decreases in tumor burden and survival advantage, respectively. These results provide a mechanistic rationale for the evaluation of LP-118 for the treatment of venetoclax responsive and relapsed CLL.

DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Transducin ß-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Leukemic B Cell CTLA-4 Suppresses Costimulation of T Cells.

The clinical benefit of CTLA-4 blockade on T cells is known, yet the impact of its expression on cancer cells remains unaddressed. We define an immunosuppressive role for tumor-expressed CTLA-4 using chronic lymphocytic leukemia (CLL) as a disease model. CLL cells, among other cancer cells, are CTLA-4+ Coculture with activated human T cells induced surface CTLA-4 on primary human CLL B cells. CTLA-4 on CLL-derived human cell lines decreased CD80 expression on cocultured CD80+ cells, with restoration upon CTLA-4 blockade. Coculture of CTLA-4+ CLL cells with CD80-GFP+ cell lines revealed transfer of CD80-GFP into CLL tumor cells, similar to CTLA-4+ T cells able to trans-endocytose CD80. Coculture of T cells with CTLA-4+ CLL cells decreased IL-2 production. Using a human CTLA-4 knock-in mouse lacking FcγR function, antitumor efficacy was observed by blocking murine CTLA-4 on tumor cells in isolation of the T cell effect and Fc-mediated depletion. These data implicate tumor CTLA-4 in cancer cell-mediated immunosuppression in vitro and as having a functional role in tumor cells in vivo.

Targeting BTK through microRNA in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Interleukin-6 in CLL: accelerator or brake?

In this issue of Blood, Li et al provide insight into the interactions between immunoreceptor signals in a human cancer microenvironment presenting a novel mechanism by which microenvironment-produced interleukin (IL)-6 acts as a tumor suppressor in chronic lymphocytic leukemia (CLL) by inhibiting toll-like receptor (TLR) signaling.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia.

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.

PKC-ß as a therapeutic target in CLL: PKC inhibitor AEB071 demonstrates preclinical activity in CLL.

Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-ß (PKC-ß) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters ß-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-ß has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted.

Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia.

The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eµ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies.

Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1.

The nuclear export receptor Exportin 1 (XPO1/CRM1) is often overexpressed in cancer cells resulting in aberrant localization of many cancer-related protein cargoes. The XPO1 inhibitor and cancer drug selinexor (KPT-330), and its analog KPT-185, block XPO1-cargo binding thereby restoring cargo localization. Selinexor binding induces cullin-RING E3 ubiquitin ligase (CRL) substrate receptor ASB8-mediated XPO1 degradation. Here we reveal the mechanism of inhibitor-XPO1 engagement by CRL5ASB8. Cryogenic electron microscopy (cryo-EM) structures show ASB8 binding to a large surface of selinexor/KPT-185-XPO1 that includes a three-dimensional degron unique to the drug-bound exportin. The structure explains weak XPO1-ASB8 binding in the absence of selinexor/KPT-185 that is unproductive for proteasomal degradation, and the substantial affinity increase upon selinexor/KPT-185 conjugation, which results in CRL5 ASB8 -mediated XPO1 ubiquitination. In contrast to previously characterized small molecule degraders, which all act as molecular glues, selinexor/KPT-185 binds extensively to XPO1 but hardly contacts ASB8. Instead, selinexor/KPT-185 binds XPO1 and stabilizes a unique conformation of the NES/inhibitor-binding groove that binds ASB8. Selinexor/KPT-185 is an allosteric degrader. We have explained how drug-induced protein degradation is mediated by a CRL5 system through an allosteric rather than a molecular glue mechanism, expanding the modes of targeted protein degradation beyond the well-known molecular glues of CRL4.

The multi-CDK inhibitor dinaciclib reverses bromo- and extra-terminal domain (BET) inhibitor resistance in acute myeloid leukemia via inhibition of Wnt/ß-catenin signaling.

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/ß-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of ß-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

CD37 in acute myeloid leukemia: a novel surface target for drug delivery.

Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes which limits broad applicability of targeted therapy. We discovered that the hematopoietic restricted tetraspanin CD37 is expressed on all primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared to normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody drug conjugate (ADC) Debio 1562 (αCD37-DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that αCD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as αCD37-DM1 is a feasible and promising therapeutic option for the treatment of AML.

Acute Lymphoblastic Leukemia with Myeloid Mutations Is a High-Risk Disease Associated with Clonal Hematopoiesis.

Myeloid neoplasms arise from preexisting clonal hematopoiesis (CH); however, the role of CH in the pathogenesis of acute lymphoblastic leukemia (ALL) is unknown. We found that 18% of adult ALL cases harbored TP53, and 16% had myeloid CH-associated gene mutations. ALL with myeloid mutations (MyM) had distinct genetic and clinical characteristics, associated with inferior survival. By using single-cell proteogenomic analysis, we demonstrated that myeloid mutations were present years before the diagnosis of ALL, and a subset of these clones expanded over time to manifest as dominant clones in ALL. Single-cell RNA sequencing revealed upregulation of genes associated with cell survival and resistance to apoptosis in B-ALL with MyM, which responds better to newer immunotherapeutic approaches. These findings define ALL with MyM as a high-risk disease that can arise from antecedent CH and offer new mechanistic insights to develop better therapeutic and preventative strategies. SIGNIFICANCE: CH is a precursor lesion for lymphoblastic leukemogenesis. ALL with MyM has distinct genetic and clinical characteristics, associated with adverse survival outcomes after chemotherapy. CH can precede ALL years before diagnosis, and ALL with MyM is enriched with activated T cells that respond to immunotherapies such as blinatumomab. See related commentary by Iacobucci, p. 142.

The role of phosphatidylinositol 3-kinase-δ in the immunomodulatory effects of lenalidomide in chronic lymphocytic leukemia.

In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.

Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.