ABC-DLBCL Cell Research Articles

TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

Ktx-120, a Novel Irakimid Degrader of IRAK4 and IMiD Substrates Shows Preferential Activity and Induces Regressions in MYD88-Mutant DLBCL CDX and PDX Models

An important goal of cancer therapy is to improve patient outcomes by driving to deep and durable tumor responses. The activity of single-agent targeted therapies, such as BTK inhibitors or IMiDs alone, has been modest in relapsed and refractory DLBCL, necessitating the use of combination therapy. Targeted protein degraders are heterobifunctional small molecules that co-opt the endogenous ubiquitin-proteasome system to drive the selective degradation of target proteins. Degraders of IRAK4, a key component of the myddosome complex, show potent and selective IRAK4 degradation and preferential activity in MYD88-mutant (MYD88MT) DLBCL models. Notably, the activity of IRAK4 degradation is superior to IRAK4 kinase inhibition, supporting the essential scaffolding role of IRAK4 in myddosome signaling. We have previously described IRAKIMiDs, novel IRAK4 degraders that utilize an IMiD as a pharmacologically active cereblon binder. These degraders simultaneously degrade both IRAK4 and IMiD substrates and show synergistic antitumor activity over either IRAK4 degraders or IMiDs alone, enabling a therapeutically relevant biological combination within a single small molecule. Here we describe KTX-120, a novel IRAKIMiD development candidate. KTX-120 is an equipotent degrader of both IRAK4 and the IMiD substrates Ikaros and Aiolos in lymphoma model systems with low single-digit nM DC50 for degradation of all substrates. The cell activity of KTX-120 has shown a high dependence on MYD88MT status: across a panel of MYD88MT cell lines, KTX-120 showed consistent and potent cell activity, with IC50 ranging from 7-29nM, whereas in MYD88WT lines, cell activity was poor ranging from 1800-3400nM. In the OCI-Ly10 ABC DLBCL cell line that harbors a MYD88L265P mutation, the cell activity of KTX-120 was associated with degradation of both IRAK4 and Ikaros, supporting the combined IRAK4 and IMiD targeting of this molecule as contributing to cell activity. Notably, the onset of cell death in OCI-Ly10 cells with KTX-120 treatment was rapid, with cells becoming committed to cell death within 72h of exposure, suggesting that continuous exposure to KTX-120 may not be necessary for antitumor activity. We explored the pharmacological activity of KTX-120 in several in vivo model systems. KTX-120 is orally bioavailable and shows dose-proportional exposure in several species. A single oral dose of KTX-120 (10 mg/Kg or 30 mg/Kg) showed significant degradation of both IRAK4 and Ikaros in a dose and time-dependent manner, with degradation of both substrates being sustained for >96h, further supporting the potential for intermittent dosing. To assess this, we have explored the antitumor efficacy of KTX-120 in intermittent dosing schedules. KTX-120 was well tolerated and showed potent antitumor activity in several CDX models of MYD88MT DLBCL, including OCI-Ly10, TMD8 and SUDHL2, achieving regressions in all models in as little as once every 2 weeks. As an example, in OCI-Ly10, a 30mpk dose Q2W drove >80% degradation of both IRAK4 and Ikaros and showed regressions (including CR) by D28. Similar activity and tolerability were seen with both PO and IV dosing at doses that achieve active exposure, enabling the potential for both oral and parenteral dosing. We have further explored the activity of KTX-120 in a collection of DLBCL patient derived xenograft models. KTX-120 shows robust activity (>85% TGI) in 4/5 models of MYD88MT DLBCL and shows no or modest activity in 2/2 models of MYD88WT DLBCL, supporting the preferential activity of this mechanism in patient samples harboring MYD88 activating mutations. Importantly, activity was observed in models with a variety of co-mutations that activate the NFkB pathway, including alterations in, CD79B and TNFAIP3, suggesting that KTX-120 has the potential for activity in MYD88MT lymphoma regardless of other mutations. Collectively, these data support the combined IRAK4 degradation and IMiD activity of KTX-120 has the potential to achieve robust and durable regressions in MYD88MT lymphomas with the increased convenience of an intermittently administered single agent and decreased potential for drug combination challenges. Disclosures Walker: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chen:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhaduri:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Nx-2127, a Degrader of BTK and IMiD Neosubstrates, for the Treatment of B-Cell Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, such as chronic lymphocytic leukemia (CLL). Covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, while effective, have been associated with the occurrence of resistance mutations. The most prevalent site of mutation, C481, renders covalent BTK inhibitors unable to form a covalent bond with BTK leading to diminished efficacy and disease progression. Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element ("harness"), a linker, and a target binding element ("hook"). NX-2127 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. NX-2127 degrades 50% of cellular BTK (DC50) at < 5 nM across multiple cancer cell lines and in human PBMCs. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 15 nM after 72 hours). Importantly, NX-2127 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-2127 EC50 values of < 30 nM versus > 1 μM for ibrutinib). Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to <10% of baseline levels at doses as low as 1 mg/kg. In addition to potent BTK degradation, NX-2127 possesses IMiD-like properties through the design of the CRBN binding harness that catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1). This activity is associated with increased T cell activation and anti-tumor effects of the IMiD drugs lenalidomide and pomalidomide. In primary human T cells, NX-2127 catalyzes the degradation of Aiolos and Ikaros with of 25 nM and 54 nM, respectively, potencies which are similar to those of lenalidomide (20 nM and 343 nM, respectively). Corresponding with such degradation, NX-2127 stimulates T cell activation as measured by increased IL-2 production in primary human T Cells in a manner similar to lenalidomide and pomalidomide. The dual activity of BTK degradation combined with immunomodulation of NX-2127 supports its development for the treatment of B-cell malignancies. Disclosures Robbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McIntosh:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wu:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kato:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Peng:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihalic:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tung:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kolobova:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Borodovsky:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ye:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Basham:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Powers:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company.

BCL10 Gain-of-Function Mutations Aberrantly Induce Canonical and Non-Canonical NF-Kb Activation and Resistance to Ibrutinib in ABC-DLBCL

ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment. Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function. BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion. To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms. As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants. This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients. Disclosures Fontan: Johnson & Johnson: Current Employment. Melnick:Epizyme: Consultancy; Jubilant: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding.

Synergistic Antitumor Effect and Mechanism of Chidamide Combined with Gemcitabine, Oxaliplatin or Zanubrutinib in Diffuse Large B-Cell Lymphoma

Aims: To observe the synergistic antitumor effect of chidamide (a novel orally benzamide class of histone deacetylase inhibitors, HDACi) combined with gemcitabine, oxaliplatin or zanubrutinib (a second generation Bruton's tyrosine kinase (BTK) inhibitor) in diffuse large B-cell lymphoma cell lines and explore the possible mechanisms. Methods: We used CCK-8 assay to test the inhibitory efficacy of single agent chidamide, gemcitabine, oxaliplatin, zanubrutinib and their combinations on DLBCL cell lines, respectively. And the combination index (CI) was calculated by the CalcuSyn software. The apoptosis and the cell cycle analysis of DLBCL cell lines under different treatments were assessed by Annexin V-APC/PI double-staining and PI staining using flow cytometry, respectively. The expression levels of acetylated histone and apoptosis-related proteins were detected by Western Blot. Results:Single-agent chidamide, gemcitabine and oxaliplatin caused growth arrest of the DLBCL cell lines (OCL-LY3、OCL-LY19、U2932、DOHH2) in a dose and time dependent manner, respectively(Figure1a,1b,1c). Single-agent zanubrutinib showed antitumor activity in two activated B-cell like DLBCL (U2932 and OCI-LY3) cell lines in a dose and time dependent manner while no antitumor activity in other two GCB-DLBCL (OCI-LY19 and DOHH2) cell lines (Figure1d). Chidamide combined with gemcitabine or oxaliplatin synergistically inhibited the proliferative viability of DLBCL cell lines. Also, chidamide could enhance the inhibitory effect of zanubrutinib in the two ABC-DLBCL cell lines. And the CI values of all groups were more than 1(Fig2-1~2-8). Chidamide could significantly increase the proportion of DLBCL cells in the G0/G1 phase in a dose-dependent manner(Figure 3). And chidamide combined with gemcitabine can synergistically enhance the cell cycle arrest in U2932 cells (Figure 4). Cell apoptosis rate increased along with the ascending dosage of chidamide in the four DLBCL cell lines. The effect of cell apoptosis became more obviously when combined with oxaliplatin or zanubrutinib (Figure 5.1~5.4). Chidamide significantly up-regulated the levels of acetylated H3 (Lys 9) and cleaved-caspase 3 while down-regulated Bcl-2 (Figure 6~7). Furthermore, PD-L1 protein was upregulated in chidamide-exposed DLBCL cell lines (Figure 8). Conclusion:Chidamide combined with gemcitabine, oxaliplatin or zanubrutinib synergistically inhibited the proliferative viability and cell apoptosis rate in DLBCL cell lines. Therefore, it is worthy to further explore the value of Chidamide combination therapy in DLBCL. Figure Disclosures No relevant conflicts of interest to declare.

Abstract PO-23: Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase

Abstract Somatic hypermutation (SHM) is a B cell-specific process that supports the affinity maturation of antibodies. The reaction targets the immunoglobulin loci (“on-target" activity) and is initiated by activation-induced cytidine deaminase (AID). AID is known to be able to mutate the genome more broadly (“off-target” activity) and hence was found to be linked to various types of B-cell malignancies. However, the mechanism by which AID is recruited and exerts its function is partially known. To unravel this puzzle, we have analyzed two types of diffuse large B-cell lymphoma (DLBCL) cell lines: germinal B cell like (GCB-DLBCL), in which AID is expressed and introduces mutations, and activated B cell like (ABC-DLBCL), where AID levels are typically increased but paradoxically, SHM is much reduced. To begin to accomplish this goal, we have taken advantage of a Diversification Activator (DIVAC)-GFP reporter assay to measure targeted SHM activity. We first confirmed that AID is expressed in ABC-DLBCL cell lines at levels up to fourfold higher than in GCB-DLBCL cell lines by RT-qPCR. In contrast to the high levels of AID seen in ABC-DLBCL cells, we have observed that SHM is reduced by as much as 10-fold in ABC versus GCB lines using the DIVAC–GFP assay. As this assay should provide a good surrogate for SHM activity at Ig loci, our data support the possibility that the Ig-locus-specific SHM targeting program is defective in ABC-DLBCL. To search for factors that might explain the differential activity of the SHM program in GCB versus ABC cells, we performed RNA-seq using cell lines from each group. Initial analysis revealed that expression of two cell surface markers, CD27 and CD38, is highly correlated with the high mutation phenotype. Flow cytometry analysis for these two markers along with the pan B cell marker CD20 revealed various subpopulations in some DLBCL lines. Isolation, characterization, and measurement of SHM activity using the DIVAC-GFP assay of these subpopulations further strengthened the correlation between CD27/CD28 expression and SHM activity. These analyses also uncovered SHM-permissive subpopulations in ABC lines and SHM-resistant subpopulations in GCB lines. This observation explains residual SHM activity in ABC lines and argues for heterogeneity in the activity of the SHM program in DLBCL cell lines and perhaps within tumor populations in patients. Future studies will include deletion and introduction of specific factors from GCB or ABC lines to validate phenotype and identify regulators of the SHM program. Finally, sequence-analysis will be done to quantify mutations with the broad goal of understanding how AID targeting and mis-targeting are regulated. Citation Format: Huseyin Saribasak, Ravi Dinesh, Lizhen Wu, David G. Schatz. Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-23.

Discovery of CA-4948, an Orally Bioavailable IRAK4 Inhibitor for Treatment of Hematologic Malignancies.

Small molecule potent IRAK4 inhibitors from a novel bicyclic heterocycle class were designed and synthesized based on hits identified from Aurigene's compound library. The advanced lead compound, CA-4948, demonstrated good cellular activity in ABC DLBCL and AML cell lines. Inhibition of TLR signaling leading to decreased IL-6 levels was also observed in whole blood assays. CA-4948 demonstrated moderate to high selectivity in a panel of 329 kinases as well as exhibited desirable ADME and PK profiles including good oral bioavailability in mice, rat, and dog and showed >90% tumor growth inhibition in relevant tumor models with excellent correlation with in vivo PD modulation. CA-4948 was well tolerated in toxicity studies in both mouse and dog at efficacious exposure. The overall profile of CA-4948 prompted us to select it as a clinical candidate for evaluation in patients with relapsed or refractory hematologic malignancies including non-Hodgkin lymphoma and acute myeloid leukemia.

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting theUPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

Abstract 4206: EUD-GK-001 is a novel kinase inhibitor with in vitro anti-lymphoma activity

Abstract Introduction. EUD-GK compounds, and in particular EUD-GK-91, show selectivity for a restricted number of kinases (ALK, RET, TRK, DDR2, ROS1) and have shown anti-tumor activity in specific solid tumor models (Zuccotto et al, EORTC-NCI-AACR 2016). Here, we have assessed a novel derivative of this class of compounds, EUD-GK-001, in a panel of lymphoma models. Methods. Lymphoma cell lines, including both B- and T-cells lymphomas, were exposed to a large range of concentrations of EUD-GK-001 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell cycle were assessed in four cell lines (FARAGE, DOHH2, TMD8, OCI-LY-10) treated with 500 nM of EUD-GK-001 for 72h. Baseline protein expression was assessed by mass spectrometry. Baseline gene expression was obtained by using the Illumina HumanHT 12 Expression BeadChips. Results. EUD-GK-001 presented a median IC50 of 1.65 μM and 3.75 μM across 14 B- and 4 T- cell lines, respectively. Five out of 14 DLBCL cells lines, including both ABC- and GCB- subtypes, presented IC50 values lower than 1 μM, A dose-depended increase of the cells in the sub-G0 phase (15-55%) was observed in two GCB-DLBCL and two ABC-DLBCL cell lines after EUD-GK-001 exposure for 72h. Taking advantage of available omics data for the tested cell lines (Cascione at al, AACR 2019), functional annotation of RNA and protein levels showed that TNF/NFKB, inflammation, IL6/JAK/STAT3, IL2/STAT5 signatures were enriched among the transcripts more expressed in the sensitive cell lines, while E2F, DNA repair, MYC signatures were enriched in the resistant cell lines. No genes coding for kinases were more expressed in both GCB (n.=2) and ABC (n=2) sensitive than in resistant DLBCL cell lines (GCB, n=2; ABC, n=3 R). However, three kinases, namely SRC, JAK2 and LCK, were significantly more expressed in EUD-GK-001 GCB-DLBCL sensitive cell lines (DOHH2, FARAGE) than in EUD-GK-001 resistant cells (SU-DHL-8, TOLEDO) and merging the top-50 mRNA and top-50 proteins identified by limma, SRC appeared as a central hub of the top genes associated with sensitivity to EUD-GK-001. The pattern of activity of EUD-GK-001 was however different from what reported for the SRC inhibitor dasatinib in the same lymphoma models (Scuoppo et al, PNAS 2019). Conclusion. EUD-GK-001 is a novel small molecule with in vitro anti-tumor activity in subsets of lymphoma models, which needs to be further explored. Citation Format: Lorenzo Scalise, Chiara Tarantelli, Fabio Zuccotto, Filippo Spriano, Michele Mascia, Luciano Cascione, Eugenio Gaudio, Mauro Angiolini, Francesco Bertoni. EUD-GK-001 is a novel kinase inhibitor with in vitro anti-lymphoma activity [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 4206.

Reimagining petroleum refining.

Rigid polymer membranes separate crude oil components without distillation

The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines.

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin) is currently being evaluated in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL). Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in two resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to RIT, alongside topoisomerase and histone deacetylases (HDAC) inhibitors.

A Novel HCK and BTK Dual Inhibitor Kin-8194 Shows Superior Activity over Ibrutinib and Overcomes BTKC481S Mediated Ibrutinib Resistance in Vitro and In Vivo in MYD88 Mutated B-Cell Lymphomas

Activating mutations in MYD88 promote malignant cell growth and survival through multiple pathways that include BTK and HCK. HCK is transcriptionally upregulated and activated by mutated MYD88 and in turn activates BTK itself, as well as ERK and AKT. Ibrutinib is a covalent inhibitor that binds to BTKCys481 and shows activity in MYD88 mutated B-cell malignancies, including WM, MZL, ABC DLBCL, and PCNSL. Resistance to ibrutinib on the basis of BTKCys481 as well as downstream mutations is increasingly being recognized. We therefore sought to develop potent and selective inhibitors that target HCK. We developed a non-covalent dual inhibitor, KIN-8194, of HCK and BTK following a screen >220 clinical and preclinical kinase inhibitors, and lead optimization following synthesis >400 analogs. Dual HCK and BTK inhibition was confirmed by both KINOMEscan® and live-cell target engagement studies (KiNativ™ profiling and live cell ATP-biotin competition assay). KIN-8194 showed robust suppression of HCK (IC50<0.495nM) and BTK (IC50=0.915nM) in these studies, and blocked pHCK and pBTK in both wild-type and mutated BTKCys481 WM and ABC DLBCL cells, and primary WM cells. Importantly, KIN-8194 showed selective killing of MYD88 mutated WM and ABC DLBCL cells and overcame resistance to ibrutinib in WM and ABC DLBCL cells engineered to express mutated BTKCys481. KIN-8194 showed excellent microsomal stability across multiple species including human (T1/2=49.5 minutes). Pharmacokinetic studies in mice showed excellent bioavailability (F=55%), serum half-life (T1/2=15.1 hours) and drug clearance (CL=17.4mL/min/kg) amenable to once daily oral dosing. The compounds exhibited an excellent in vitro safety profiling including no relevant inhibition observed against a panel of 100 other receptor targets, including hERG, AMES was negative up to 50 µM, and Cyp inhibition studies showed acceptable inhibition up to 10 µM. Pharmacodynamic studies following oral administration showed that KIN-8194 blocked both pHCK and pBTK in wild-type and mutated BTKCys481 expressing TMD8 ABC DLBCL cells engrafted in NOD SCID mice. Continuous dosing up to 100 mg/kg was well tolerated in these mice. KIN-8194 treated NOD SCID mice xenografted with either wild-type (A) or mutated (B) BTKCys481 TMD cells (N=8/cohort) showed superior tumor growth suppression and survival over vehicle control or ibrutinib treated mice at 50 mg/kg. Among wild-type BTKCys481 TMD8 xenografted mice treated for 6 weeks, elimination of tumor was observed in half the mice with no subsequent growth following 12 additional weeks of observation, consistent with a cure. We therefore describe a novel, highly potent non-covalent dual HCK and BTK inhibitor that is well tolerated in mice, shows selective killing of MYD88 mutated WM and ABC DLBCL cells, and can overcome mutated BTKCys481 related ibrutinib resistance. Disclosures Hunter: Janssen: Consultancy. Castillo:Beigene: Consultancy, Research Funding; TG Therapeutics: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, 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. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

Curcumin Enhances the Sensitivity of Bortezomib By Inhibiting NF-Κb in ABC Diffuse Large B-Cell Lymphoma Cells

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. According to gene expression profile, DLBCL can be divided into at least two subtypes: germinal center B cell type (GCB) and activated B cell type (ABC). ABC-type DLBCL is commonly activated by the NF-κB pathway, but the proteasome inhibitor bortezomib does not significantly improve the prognosis of ABC-DLBCL. Curcumin inhibit the proliferation of various tumor cells by inhibiting the activation of NF-κB. Our purpose was to evaluate whether curcumin could enhance the ensitivity of Bortezomib in ABC-DLBCL. Methods MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with curcumin and bortezomib. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green.Western Blot was used to evaluated the expression of PARP, NF-κB, I κBα/p-IκBα and caspase-3 in ABC-DLBCL cells treated with curcumin and bortezomib. Results Both curcumin and bortezomib could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. Curcumin decreased the expression of p-IκBα, NF-κB/p65 and increased the expression of Cleaved PARP in ABC-DLBCL cell lines. Caspase Inhibitor Z-VAD could reverse the curcumin induced proliferation inhibition and apoptosis by decreasing the cleaved PARP expression. The combination of curcumin and bortezomib could further enhance the proliferation inhibition and apoptosis in ABC-DLBCL cell lines by inhibiting NF-κB. Conclusions Curcumin induced proliferation inhibition and apoptosis by inhibiting NF-κB in ABC-DLBCL. It may sensitize ABC-DLBCL cell lines to the cytotoxic effects of bortezomib by inhibiting NF-κB. Disclosures No relevant conflicts of interest to declare.

Chemically Induced Degradation of MALT1 to Treat B-Cell Lymphomas

MALT1 is a protease and scaffold protein involved in signal transduction to NF-κB downstream of several receptors including B-cell (BCR) and T-cell receptors (TCR). MALT1 is aberrantly activated in ABC DLBCL by mutations in upstream genes in the BCR and TLR pathways (CD79A/B, CARD11, MYD88) and is critical for proliferation and survival. Recent studies by our lab, and others, identified inhibitors of MALT1 protease activity that revealed MALT1 is therapeutically targetable in ABC DLBCL. MALT1 is also essential for CLL, MCL and certain solid tumors (most notably lung cancer and glioblastoma). A first-in-man clinical trial recently started to evaluate MALT1 protease inhibition in B-cell non-Hodgkin's lymphomas. However, chronic inactivation of MALT1 protease activity suppressed T regulatory cells in vivo in protease dead murine models causing fast progressing autoimmune disease and death. Loss of MALT1, on the other hand, has also potent anti-tumoral effects but does not lead to autoimmunity in murine models. These findings prompted us to study alternative MALT1 targeting therapeutic approaches aimed to target its scaffolding activity. We have developed a series of proteolytic targeting chimera (PROTAC) compounds against MALT1. PROTACs are bifunctional compounds that induce selective proteolysis by targeting proteins of interest to E3 ligases for directed proteosomal degradation. Our MALT1 PROTACs are based on an allosteric MALT1 inhibitor that binds reversibly to MALT1 and fused to a Cereblon (CRBN) binding moiety, with the intent of bringing the CRBN E3 ligase complex in close proximity of MALT1 and promoting its ubiquitination and proteasomal degradation. We performed structure-activity relationship analysis and studied: 1) three linker attachment points in the MALT1 binding moiety and evaluated their MALT1 enzymatic inhibitory activity and binding to MALT1; 2) the effect of distinct linker length and polarity in MALT1 degradation and, 3) compared the effect of Lenalidomide, Pomalidomide and CC-220, which have increasing CRBN affinities, as alternative CRBN-binding moieties. Six out of eighteen compounds presented higher than 50% MALT1 degradation at 1 μM compared to vehicle treated cells in a MALT1-dependent cell line, OCI-Ly3. The parental allosteric compound, on the other hand, did not affect MALT1 levels compared to vehicle treated cells and was used as a negative control for MALT1 degradation. Compounds that actively degraded MALT1 over 50% preserved selective killing of ABC DLBCL over GCB DLBCL, same as the parental MALT1 inhibitor. GI50 of active compounds in OCI-Ly3 was 2-6 μM while it was greater than 20 μM for the MALT1-independent cell line OCI-Ly1. We chose two of our most effective and selective compounds to validate MALT1 PROTACs mechanism of action. Unlike their parental MALT1 targeting allosteric compound, MALT1 PROTACs effectively degraded MALT1 in a CRBN-dependent manner as shown in a 293T-CRBN knockout cell line or in OCI-Ly3 cells by treatment with 1 μM MLN4924. MLN4924 inhibits NEDD8-activating enzyme which is essential for the CRBN complex to function. Notably, MALT1 PROTACs degraded MALT1 in OCI-Ly1 cells (FC=-2.5) and Raji cells (FC=-1.7), where MALT1 is inactive. MALT1 degradation by PROTACs was not affected by activation in Raji cells, since PMA/ionomycin treatment did not alter the effect of MALT1 PROTACs on MALT1 levels. Therefore, MALT1 PROTACs can degrade MALT1 independent of its activation state. Moreover, unlike MALT1 protease inhibitors, MALT1 PROTACs potently suppress NF-κB activation, which is dependent on MALT1 scaffolding activity, as assessed by WB of phopho and total IκB in ABC DLBCL cell lines. Our data shows that MALT1 PROTACs could be excellent agents for the treatment of ABC DLBCL and other lymphomas, providing an alternative to enzymatic targeting that might prove useful to avoid autoimmunity or overcome resistance mechanisms. Disclosures 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. Melnick:Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

Targeted Protein Degradation of BTK As a Unique Therapeutic Approach for B Cell Malignancies

Bruton's tyrosine kinase (BTK) is a key component of B cell receptor signaling and is involved in B cell development and function. BTK plays a crucial role in cell survival in B cell malignancies such as Chronic Lymphocytic Leukemia (CLL), and covalent inhibitors of BTK, such as ibrutinib, have been successful clinically. However, long-term therapy with covalent BTK inhibitors has been shown to generate resistance mutations, which lead to disease progression. New treatments are needed to address this unmet medical need. Small molecule-induced protein degradation offers a unique approach to inhibiting BTK function. Chimeric Targeting Molecules (CTMs) mediate ubiquitylation and proteasomal degradation of specific target proteins. CTMs are comprised of a ubiquitin ligase binding element ("harness"), a chemical linker, and a target binding element ("hook"). Use of CTMs to degrade both WT and ibrutinib-resistant forms of BTK present a novel approach to targeting BTK and could affect both its catalytic and potential scaffolding functions. We have identified multiple CTMs that catalyze BTK degradation in multiple B cell lines; the concentration of one of such CTM, NRX0492, required to degrade 50% BTK (DC50) was < 1 nM after 4 hours. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 10 nM after 72 hours). These CTMs also induce degradation of the ibrutinib-resistant C481S mutant form of BTK in cells and confer loss of viability in BTKC481S mutant TMD8 cells with EC50 values of < 10 nM compared to > 1 µM for ibrutinib. Oral administration of NRX0492 in mice leads to dose-proportional exposure in plasma and BTK degradation in circulating and splenic B cells: at 6 hours after a single oral dose of NRX0492, 11% BTK remained in mouse splenocytes compared to 100% BTK in mice dosed with vehicle (P < 0.0001). In a WT TMD8 xenograft model, NRX0492 treatment resulted in similar tumor growth inhibition (TGI) as compared to ibrutinib over 23 days of daily oral administration: 54.4% TGI for NRX0492 and 55.8% for Ibrutinib, both as compared to placebo (P = 0.0006 and P = 0.0004, respectively). Notably, in a TMD8 BTKC481S xenograft model, NRX0492 demonstrated superior TGI as compared to ibrutinib: 51.3% versus 15.2%, (P = 0.033). Preclinical safety and toxicity studies for BTK CTMs are ongoing to inform plans for clinical development. CTM-mediated degradation of BTK may provide an alternative therapeutic approach for B cell malignancies, particularly in the ibrutinib-resistant setting. Disclosures Kelly: Nurix Therapeutics: Employment. Robbins:Nurix Therapeutics: Employment. Tan:Nurix Therapeutics: Employment. Tenn-McClellan:Nurix Therapeutics: Employment. McIntosh:Nurix Therapeutics: Employment. Wu:Nurix Therapeutics: Employment. Konst:Nurix Therapeutics: Employment. Kato:Nurix Therapeutics: Employment. Perez:Nurix Therapeutics: Employment. Tung:Nurix Therapeutics: Employment. Kolobova:Nurix Therapeutics: Employment. Ingallinera:Nurix Therapeutics: Employment. McKinnell:Nurix Therapeutics: Employment. Weiss:Nurix Therapeutics: Employment. Noviski:Nurix Therapeutics: Employment. Ye:Nurix Therapeutics: Employment. Peng:Nurix Therapeutics: Employment. Cardozo:Nurix Therapeutics: Employment. Mihalic:Nurix Therapeutics: Employment. Basham:Nurix Therapeutics: Employment. Rountree:Nurix Therapeutics: Employment. Karr:Nurix Therapeutics: Employment. Bence:Nurix Therapeutics: Employment. Zapf:Nurix Therapeutics: Employment. Sands:Nurix Therapeutics: Employment.

The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37 Targeted Radioimmunotherapy in Activated B Cell like Diffuse Large B Cell Lymphoma Cell Lines

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin®) is currently being evaluated as monotherapy in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL), as well as in a phase 1b trial in combination with rituximab for patients with relapsed/refractory FL. Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines: U-2932 and RIVA. Both cell lines are representative for TP53 deficient Double Expressor (DE) DLBCL. Importantly, resistance was not a consequence of reduced binding of the radioimmunoconjugate to cell surface expressed CD37. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in both resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to radioimmunotherapy (RIT), alongside topoisomerase and histone deacetylases (HDAC) inhibitors. Kinetic studies of the effect of mono- and combination therapy of U-2932 and RIVA cells with JNJ-7706621 and 177Lu-lilotomab satetraxetan are suggestive of a model in which radiation damage induced G2-arrested lymphoma cells eventually enter mitosis (repair or escape) and mitotic entry, progression and exit are impaired by JNJ-7706621 mediated inhibition of CDK1/2 and AURKA/B. Extended residence-time of cells in mitosis due to chromosome condensation and congression defects as well as spindle and mid-spindle assembly failure is likely pivotal for the increased sensitivity to persistent 177Lu-lilotomab satetraxetan deposited DNA damage, ultimately promoting cytokinesis failure (multinucleation, aneuploidy, increased cell size) and cell death. In conclusion, CD37-targeting 177Lu-lilotomab satetraxetan RIT showed activity in several ABC-DLBCL lymphoma cell lines. CD37-independent RIT-resistance was identified in two cell lines representative of aggressive DE ABC-DLBCLs with inactive TP53, and reversed by subsequent inhibition of CDK1/2 and AURKA/B by JNJ-7706621. These findings may be of potential relevance for ongoing clinical trials of 177Lu-lilotomab satetraxetan in relapsed, ASCT-non-eligible DLBCL, and may also be more generally applicable to other 177Lu-based RITs and alternative radionuclide utilizing targeted therapies. Future pre-clinical investigations are required to elucidate the potential application of CDK1/2 and AURKA/B inhibitors as a strategy to revert RIT resistance in TP53 deficient cancers. Disclosures Rødland: Nordic Nanovector ASA: Patents & Royalties, Research Funding. Melhus:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Generalov:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Bertoni:Nordic Nanovector ASA: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants; NEOMED Therapeutics 1: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties. Syljuåsen:Nordic Nanovector ASA: Patents & Royalties, Research Funding. Patzke:Nordic Nanovector ASA: Employment, Patents & Royalties.

Proteasomal cysteine deubiquitinase inhibitor b-AP15 suppresses migration and induces apoptosis in diffuse large B cell lymphoma

BackgroundThe first line therapy for patients with diffuse large B cell (DLBCL) is R-CHOP. About half of DLBCL patients are either refractory to, or will relapse, after the treatment. Therefore, identifying novel drug targets and effective therapeutic agents is urgently needed for improving DLBCL patient survival. b-AP15, a selective small molecule inhibitor of proteasomal USP14 and UCHL5 deubiquitinases (DUBs), has shown selectivity and efficacy in several other types of cancer cells. This is the first study to report the effect of b-AP15 in DLBCL.MethodsCell lines of two DLBCL subtypes, Germinal Center B Cell/ GCB (SU-DHL-4, OCI-LY-1, OCI-LY-19) and Activated B Cell/ABC (SU-DHL-2), were used in the current study. Cell viability was measured by MTS assay, proliferation by trypan blue exclusion staining assay, cellular apoptosis by Annexin V-FITC/PI staining and mitochondrial outer membrane permeability assays, the activities of 20S proteasome peptidases by cleavage of specific fluorogenic substrates, and cell migration was detected by transwell assay in these GCB- and ABC-DLBCL cell lines. Mouse xenograft models of SU-DHL-4 and SU-DHL-2 cells were used to determine in vivo effects of b-AP15 in DLBCL tumors.Resultsb-AP15 inhibited proteasome DUB activities and activated cell death pathway, as evident by caspase activation and mitochondria apoptosis in GCB- and ABC- DLBCL cell lines. b-AP15 treatment suppressed migration of GCB- and ABC-DLBCL cells via inhibiting Wnt/β-catenin and TGFβ/Smad pathways. Additionally, b-AP15 significantly inhibited the growth of GCB- and ABC DLBCL in xenograft models.ConclusionsThese results indicate that b-AP15 inhibits cell migration and induces apoptosis in GCB- and ABC-DLBCL cells, and suggest that inhibition of 19S proteasomal DUB should be a novel strategy for DLBCL treatment.

Abstract LB-272: KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition

Abstract Purpose: This work assessed the antitumor activity of selective small molecule IRAK4 degraders in human ABC DLBCL cell lines in vitro and in tumor xenograft models in vivo, alone and in combination with BTK inhibition. Introduction: ABC DLBCL comprises approximately 45% of DLBCL and has a worse outcome with R-CHOP chemotherapy compared to GCB DLBCL. Activating mutations in MYD88 occur in 30-40% of ABC DLBCL; L265P, the most prevalent MYD88 mutation, causes constitutive assembly and activation of the Myddosome. IRAK4 kinase and scaffolding functions are essential for full signaling through the Myddosome to NFκB and MAPK pathways. Kymera Therapeutics is using a chemical knockdown strategy to develop heterobifunctional small molecule IRAK4 degraders, exemplified by KYM-001, for the treatment of MYD88-driven lymphomas. Methods: IRAK4 in human PBMC, ABC DLBCL cell lines and xenografts was quantified by immunoassays or targeted MS/MS. Myddosome signaling was monitored by mRNA and phosphoprotein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume. Key data: KYM-001 led to potent E3 ligase-dependent degradation of IRAK4. Notably, KYM-001 more effectively inhibited TLR-activated Myddosome signaling compared to IRAK4 kinase inhibitors in human PBMC. Degradation was highly selective for IRAK4 vs >10,000 other detected proteins in the MYD88 L265P mutant ABC DLBCL line OCI-LY10. IRAK4 degradation by KYM-001 resulted in cell cycle inhibition and apoptosis within 48-72 h in ABC DLBCL, with preferential activity in MYD88-mutant vs MYD88-WT cell lines. Oral dosing of KYM-001 showed dose-dependent antitumor activity in several mouse xenograft models of human MYD88-mutant ABC DLBCL at tolerated doses and schedules. In the OCI-LY10 model, tumor regression was associated with >80% degradation of IRAK4, establishing the pharmacodynamic effect required for maximal efficacy. Since alterations in BCR signaling and MYD88 frequently co-occur in B-cell malignancies, we investigated the potential for combined activity of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has activating mutations in both CD79B and MYD88, BTK inhibition with ibrutinib had an additive effect on KYM-001 antitumor activity. Conclusions: KYM-001 is a first-in-class, potent, selective and orally active IRAK4 degrader that causes tumor regression in ABC-DLBCL models. Degradation of IRAK4 removes both the kinase and scaffolding functions of IRAK4, and may be superior to kinase inhibition alone. These data support IRAK4 degraders as a promising new therapeutic opportunity for MYD88-driven lymphoma, both alone and in combination with other targeted approaches such as BTK inhibition. Citation Format: Joseph F. Kelleher, Veronica Campbell, Jesse Chen, Jared Gollob, Nan Ji, Hari Kamadurai, Christine Klaus, Henry Li, Christine Loh, Alice McDonald, Haojing Rong, Scott Rusin, Kirti Sharma, Dominico Vigil, Duncan Walker, Matt Weiss, Karen Yuan, Yi Zhang, Laurent Audoly, Nello Mainolfi. KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-272.