Upregulation of MYC is a common driver event in human cancers, and some tumors depend on MYC to maintain transcriptional programs that promote cell growth and proliferation. Preclinical studies have suggested that individually targeting upstream regulators of MYC, such as histone deacetylases (HDAC) and phosphoinositide 3-kinases (PI3K), can reduce MYC protein levels and suppress the growth of MYCdriven cancers. Synergy between HDAC and PI3K inhibition in inducing cancer cell death has also been reported, but the involvement of MYC regulation is unclear. In this study, we demonstrated that HDAC and PI3K inhibition synergistically downregulates MYC protein levels and induces apoptosis in "double-hit" (DH) diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, CUDC-907, a small-molecule dual-acting inhibitor of both class I and II HDACs and class I PI3Ks, effectively suppresses the growth and survival of MYC-altered or MYC-dependent cancer cells, such as DH DLBCL and BRD-NUT fusion-positive NUT midline carcinoma (NMC) cells, and MYC protein downregulation is an early event induced by CUDC-907 treatment. Consistently, the antitumor activity of CUDC-907 against multiple MYC-driven cancer types was also demonstrated in animal models, including DLBCL and NMC xenograft models, Myc transgenic tumor syngeneic models, and MYC-amplified solid tumor patient-derived xenograft (PDX) models. Our findings suggest that dual function HDAC and PI3K inhibitor CUDC-907 is an effective agent targeting MYC and thus may be developed as potential therapy for MYCdependent cancers.

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

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The clinical success of antibody-mediated immune checkpoint blockade therapies has transformed the cancer therapy paradigm by demonstrating that durable antitumor immune responses and long-term remissions may be achieved in a subset of patients across a diverse range of cancers. However, the majority of patients fail to respond to antibody therapies targeting single immune checkpoint pathways and antibodies exhibit a long in vivo half-life (>15-20 days with >70% target occupancy for months) which may contribute to the emergence of immune-related adverse events. Additionally, antibody therapies must be administered by intravenous infusion in a hospital or clinic which places an additional burden on patients who may have mobility challenges. Thus, there is a significant opportunity for a novel immune checkpoint therapy that can address the shortcomings associated with the current antibody therapies. CA-170 is a small molecule, orally bioavailable antagonist of the PD-L1, PD-L2 and VISTA/PD-1H immune checkpoint pathways which is currently undergoing Phase I clinical testing. In preclinical safety studies conducted in rodents and non-human primates, orally administered CA-170 shows no signs of toxicity when dosed up to 1000 mg/kg for 28 consecutive days. CA-170 exhibits an oral bioavailability of approximately 40% and <10% in mouse and monkey, respectively, and the plasma half-life ranges from approximately 0.5 hours for mouse to approximately 3.25-4.0 hours for cynomolgus monkey. The ability of CA-170 to disrupt the signaling of PD-1/PD-L1/2 or VISTA/PD-1H has been inferred though in vitro functional studies. CA-170 exhibits potent activity comparable to that of blocking PD-1 or VISTA antibodies when tested in cell culture assays to rescue the proliferation or IFN-γ secretion of lymphocytes stimulated in the presence of inhibitory PD-L1, PD-L2 or VISTA/PD-1H proteins. In mice, orally administered CA-170 inhibits the growth of syngeneic tumors, enhances peripheral T cell activation, and promotes the activation of tumor infiltrating CD8+ T cells in a dose dependent manner. These non-clinical data provide a strong rational for the continued Phase I clinical development of CA-170, the first oral, small molecule immune checkpoint antagonist for the treatment of advanced cancers. Citation Format: Adam S. Lazorchak, Troy Patterson, Yueyun Ding, Pottayil G. Sasikumar, Naremaddepalli S. Sudarshan, Nagaraj M. Gowda, Raghuveer K. Ramachandra, Dodheri S. Samiulla, Sanjeev Giri, Rajesh Eswarappa, Murali Ramachandra, David Tuck, Timothy Wyant. CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A36.

MYC family genes are among the most frequently deregulated oncogenic drivers in human cancer. Pharmacologic inhibition of HDAC activity and blockade of the PI3K pathway have both been shown to suppress MYC-induced transcription. HDAC activity is critical for MYC gene regulation, as MYC represses transcription of target genes through recruitment of HDACs. HDAC inhibitors have been shown to restore expression of genes suppressed by MYC family members and to induce rapid downregulation of expression of MYC itself. The PI3K pathway plays a central role in regulating MYC at the post-transcriptional level. Activation of PI3K signaling leads to activation of AKT, which phosphorylates and inhibits GSK3β. As GSK3β normally phosphorylates MYC which facilitates the degradation of MYC, activation of PI3K signaling leads to increased stability of MYC, whereas PI3K inhibitors decrease MYC stability. A recent study has demonstrated addiction to MYC signaling and hypersensitivity to PI3K inhibition in PTEN-deficient diffuse large B-cell (DLBCL) cell lines, suggesting that MYC-driven cancers may be particularly sensitive to PI3K inhibition. As HDACs and PI3K regulate MYC protein levels and functions through nonoverlapping mechanisms, simultaneous HDAC and PI3K inhibition may further enhance MYC suppression. CUDC-907 is an orally bioavailable, small-molecule dual HDAC and PI3K inhibitor that primarily inhibits class I and II HDACs and the PI3Kα, β, and δ isoforms. CUDC-907 shows greater anti-tumor activity in vitro than single-target HDAC or PI3K inhibitors, especially in MYC-dependent cell types, such as DLBCL and NUT midline carcinoma (NMC). In preclinical testing, CUDC-907 treatment leads to a dose-dependent decrease in MYC protein levels, and is also more potent in decreasing MYC than the HDAC inhibitor panobinostat and the pan-PI3K inhibitor pictilisib alone or in combination. Significant antitumor effects have been consistently observed in MYC-driven DLBCL xenograft and genetically engineered mouse models exposed to CUDC907. In particular, certain MYC translocation (Daudi), double-hit (concurrent MYC and BLC2 translocation, WSUDLCL2 and DOHH2), double-expresser (expression of MYC and BCL2 proteins, U2932) xenograft models, and the Eμ-Myc transgenic mouse model achieve tumor growth inhibition of 100%, 69%, 56%, 97% and 72%, respectively. These findings raise the possibility that hematologic and solid tumors driven by aberrant overexpression of MYC family genes (e.g., MYC-altered DLBCL and NMC) might be more responsive to simultaneous HDAC and PI3K inhibition with CUDC-907 than they are to single-target therapy. Clinical Phase 1 studies are currently testing CUDC-907 in patients with relapsed/refractory (R/R) DLBCL and advanced MYC-aberrant solid tumors. Preliminary data are encouraging and support the planned Phase 2 study in R/R MYC-altered DLBCL, as well as further testing in other MYC-driven malignancies. Citation Format: Kaiming Sun, Ruzanna Atoyan, Mylissa A. Borek, Steven Dellarocca, Maria E. Samson, Anna W. Ma, Guangxin Xu, Troy Patterson, David P. Tuck, Jaye L. Viner, Ali Fattaey, Jing Wang. Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4634.

The T-Cell Receptor (TCR) can be functionally split into antigen recognition mediated by the TCRα/β heterodimer and signal transduction triggered by the CD3 complex comprising CD3ϵ/γ, CD3ϵ/δ and CD3ζ/ζ dimers. Unlike antibodies, the TCR recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules. Recently, we reported that tethering an antibody-derived binder to one of the TCR subunits redirects T cells to specifically kill tumor cells independent of HLA. Different from CAR-T cells, the T cell receptor fusion constructs (TRuC™) are integrated into the natural TCR and require all TCR subunits for receptor translocation to the cell surface. The development of off-the-shelf TRuC-T cells is desirable to shorten the vein-to-vein production time and reduce manufacturing costs. Here, we describe the generation of TRuC-T cells expressing a fully functional TRuC TCR without alloreactivity. Inactivation of the endogenous TRAC gene employing CRISPR/Cas9 endonuclease disrupts natural TCR formation. Yet, replacing the endogenous TCRα subunit with a human TCRα constant region without variable domain to avoid alloreactivity is insufficient for TRuC TCR expression. A functional TRuC TCR, however, can be created by substituting the following constructs for the inactivated TRAC gene: (i) murine TCRα and β constant domains without variable domains, (ii) chimeric human/murine TCRα and β constant regions or (iii) TCRγ and δ constant domains. Off-the-shelf TRuC-T cells upregulate activation markers, secrete cytokines, and kill tumor cells in an antigen-specific manner. Importantly, the genome-engineered TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft models without signs of Graft versus host disease (GvHD). Our findings warrant further development of allogeneic TRuC-T cells for cancer therapy. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Troy Patterson, Tiffany Chan, Holly Horton, Robert Tighe, Dario A. Gutierrez, Daniel Getts, Robert Hofmeister. Engineering off-the-shelf T cell receptor fusion construct (TRuC) T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2190.

Cluster of differentiation 28 (CD28) and programmed death receptor 1 (PD-1) are members of the CD28 superfamily of co-receptors that have critical roles in the regulation of T cell-mediated immunity and inflammation. Ligation of CD28 synergizes with T cell receptor (TCR) signaling to enhance T cell activation through the PI3K-Akt pathway, while PD-1 ligation by its ligands (PD-L1/L2) sequesters critical mediators of signaling from the TCR complex, thereby shunting T cell activation and effector function. Thus, the expression of PD-L1/L2 in solid tumors may pose a significant barrier to anti-tumor immunity and the efficacy of adoptive T cell therapies (ACT). We have recently described a novel class of engineered T cells that integrate a T cell receptor fusion construct (TRuC®) into the natural TCR complex, thereby reprogramming the specificity of the T cell to recognize tumor surface antigen in a human leukocyte antigen (HLA)-independent fashion. TC-210 T cells expressing mesothelin (MSLN) specific TRuCs demonstrate robust anti-tumor immunity in preclinical models of mesothelioma, protecting mice from tumor re-challenge while inducing lower levels of inflammatory cytokine release when compared to a 2nd generation MSLN-targeted CAR T. Here, we show that co-expression of a PD-1:CD28 switch receptor comprising the PD-1 extracellular domain fused to the CD28 intracellular domain, enhances the activity of TC-210 T cells. When compared to TC-210 expressing only the TRuC, co-expression of PD1:CD28 was able to restore PD-L1 mediated inhibition of cytokine production and proliferation in co-culture with tumor cells. In vivo and molecular mechanistic studies are currently underway. Citation Format: Derrick P. McCarthy, Sarah Guyette, Michael Lofgren, Jyothi Sethuraman, Thamara DeSilva, Ahmar Aziz, Troy Patterson, Shruti Datari, Tiffany Chan, Philippe Kieffer-Kwon, Christopher J. Rold, Reshma Singh, Jian Ding, Holly Horton, R. Anthony Barnitz, Andrew Cornforth, Robert Tighe, Robert J. Hofmeister, Dario A. Gutierrez. A chimeric PD1-CD28 switch receptor enhances the activity of TRuC-T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 893.

The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1514.