Purpose: Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. Experimental Design: We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. Results: Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. Conclusions: Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic approaches. Pharmacological inhibitors of cyclin-dependent kinases (CDK) demonstrated pre-clinical activity in DLBCL, however many stalled in clinical development. Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells. CDK9 inhibition (CDK9i) resulted in rapid changes in the transcriptome and proteome, with downmodulation of multiple oncoproteins (eg, MYC, Mcl-1, JunB, PIM3) and deregulation of phosphoinotiside-3 kinase (PI3K) and senescence pathways. Following initial transcriptional repression due to RNAPII pausing, we observed transcriptional recovery of several oncogenes, including MYC and PIM3. ATAC-Seq and ChIP-Seq experiments revealed that CDK9i induced epigenetic remodeling with bi-directional changes in chromatin accessibility, suppressed promoter activation and led to sustained reprograming of the super-enhancer landscape. A CRISPR library screen suggested that SE-associated genes in the Mediator complex, as well as AKT1, confer resistance to CDK9i. Consistent with this, sgRNA-mediated knockout of MED12 sensitized cells to CDK9i. Informed by our mechanistic findings, we combined AZD4573 with either PIM kinase or PI3K inhibitors. Both combinations decreased proliferation and induced apoptosis in DLBCL and primary lymphoma cells in vitro as well as resulted in delayed tumor progression and extended survival of mice xenografted with DLBCL in vivo. Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.

Introduction: Activated B cell receptor (BCR) signaling is a hallmark of NHL. BCR-associated kinases LYN, SYK, BTK and PI3K activate pro-survival signaling pathways including MEK/ERK, AKT/mTOR, and NFκB. While targeting BTK (ibrutinib, acalabrutinib) and PI3K (idelalisib, duvelisib) has shown efficacy in CLL, clinical responses fall short in aggressive NHL, necessitating the development of novel approaches to suppress BCR signaling. CG-806 is a BTK/cluster-selective kinase inhibitor currently under investigation in phase 1 clinical trials for patients with hematological malignancies. CG-806 targets both WT BTK (IC50 ~ 8 nM) and the BTKC481S (IC50 ~ 2.5 nM; www.aptose.com). Here we investigate the anti-tumor effects of CG-806 in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). Methods: CG-806 was provided by Aptose Biosciences, Inc. (San Diego, CA). DLBCL and MCL cell lines were assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), mitochondrial mass and mitophagy. Ibrutinib (ibr) resistance was induced by exposure over 6 months. Primary peripheral blood mononuclear cells were incubated for 24 h in media conditioned by stromal cells engineered to express CD40L or BAFF prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected into the tail vein of NSG mice and tracked weekly by flow cytometry (CD5+ CD19+ CD45+). Upon MCL detection in the peripheral blood, mice began daily treatment with 30.8 or 308 mg/kg CG-806 or vehicle control via oral gavage until moribund. Splenocytes were harvested 1 h after the final drug treatment. Results: CG-806 potently inhibited proliferation of both parental and ibr-resistant MCL cell lines (Mino, JeKo-1) with IC50<0.01 μM at 72 h. DLBCL cell lines (U2932, OCI-LY3 OCI-LY19) demonstrated moderate sensitivity to CG-806 (IC50 0.3-1 μM), while SU-DHL10 was highly sensitive (IC50<0.01 µM). Treatment with CG-806, but not ibrutinib, induced apoptosis of primary MCL cells in CD40L- or BAFF-expressing stromal co-cultures. Following anti-IgM crosslinking of primary cells, treatment with CG-806 decreased phosphorylation of SYK, BTK, AKT and ERK, indicating disrupted BCR signaling. Treatment with CG-806 increased respiratory reserve capacity but did not impact the basal oxygen consumption rate in both parental and ibr-resistant MCL cell lines. Basal extracellular acidification rate (ECAR) was increased following CG-806 treatment, indicating heightened glycolytic activity. Furthermore, CG-806-treated cells demonstrated potent induction of mitophagy accompanied by a reduction in mitochondrial mass. CG-806 slowed expansion of circulating MCL cells and reduced proliferation of spleen-resident MCL cells in both chemo- and ibr-resistant MCL PDX models. CG-806 and ibrutinib extended survival of chemoresistant PDX mice without evidence of toxic events. Treatment with CG-806 led to decreased phosphorylation of SYK, BTK, and AKT but also upregulated expression of BCL2 and BCLX. RNA-seq analysis of spleen-resident cells revealed downregulation of NFκB targets and JAK/STAT signaling in ibr-resistant PDX mice treated with CG-806. This was accompanied by enrichment of metabolic pathways (oxidative phosphorylation, fatty acid metabolism) and MYC targets. Next, we evaluated CG-806 for synthetic lethality in a functional in vitro screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). Consistent with the above observations, synergy was observed between CG-806 and inhibitors of metabolic enzymes (teleglenastat, perhexiline maleate) and BH3-mimetics targeting BCL2/X proteins (venetoclax, AZD4320). Conclusions: Our data demonstrate preliminary efficacy of CG-806 in MCL and DLBCL in vitro and in MCL DPX models. CG-806 treatment led to metabolic reprograming towards glycolysis and induced mitophagy. BCL2 family proteins may be implicated in resistance to CG-806. These results provide rationale for further investigation of CG-806 in aggressive NHL. Disclosures Tyner: Array: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Agios: Research Funding; Aptose: Research Funding. Danilov:Pharmacyclics: Consultancy; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; Celgene: Consultancy; Aptose Biosciences: Research Funding; Bristol-Myers Squibb: Research Funding; Rigel Pharmaceuticals: Consultancy; Karyopharm: Consultancy; BeiGene: Consultancy; Abbvie: Consultancy.

Introduction Oncogenic programs are facilitated by activators of transcriptional machinery, including certain CDKs. CDK9, a component of the positive transcription elongation factor b (pTEFb) complex, has arisen as an attractive target due to its regulation of MYC and MCL1 transcription (Hashiguchi et al, 2019). Nevertheless, we and others have observed resistance to CDK9i in vitro and in vivo. Here we studied the effects of CDK9 inhibition using the novel selective CDK9 inhibitor AZD4573, currently under evaluation in clinical trials. Methods A panel of NHL cell lines (OCI-LY3/19, SUDHL4/10/16, VAL, U2932) and primary NHL cells were employed. Response to CDK9i was characterized using LC-MS proteomic analysis, RNA-Seq, and CRISPR-Cas9 Screening. Results NHL cells treated with AZD4573 for 6h exhibited a dose dependent reduction in phospho-RNAPIISer2, as well as loss of MYC and Mcl-1. CDK9i potently inhibited proliferation and induced apoptosis in a panel of NHL cell lines (IC50 range 5-30 nM). Two DLBCL cell lines underwent LC-MS proteomic analysis following AZD4573 treatment (30 nM, 3h). Treated cells exhibited rapid loss of MYC, Mcl-1, PIM3 and JUNB protein levels. We observed broad transcriptional repression via RNA-seq, including downregulation of PIM3 and JUNB (30 nM, 3h). However, a subset of genes, including MYC, PIM1 and JUNB underwent early transcriptional recovery, confirmed by immunoblotting, thus identifying candidate genes which may account for resistance to CDK9i. PIM kinases cooperate with the PI3K/ATK signaling pathway, and have been proposed as therapeutic targets in cancer. We next used SGI1776 (PIM1 specific) and AZD1208 (pan-PIM) in combination with AZD4573, and found synergy between them in a panel of 4 cell lines and primary samples. OCI-LY3 xenograft mice treated with a combination of AZD4573 (15 mg/kg; IP; once weekly) and AZD1208 (30 mg/kg; oral gavage, twice weekly) demonstrated restricted tumor growth and increased survival compared to control. To further understand pathways mediating resistance to CDK9i, we carried out a genome-wide loss of function CRISPR-Cas9 library screen. Two Cas9-expressing NHL cell lines were transduced with a CRISPR library comprised of ~5 unique sgRNA per gene. Loss of AKT, RPTOR, or mTOR, among others, sensitized cells to AZD4573. Concurrent treatment with PI3K inhibitors synergistically suppressed proliferation of NHL cell lines and primary cells treated with AZD4573 in vitro. OCI-LY3 xenograft mice were treated with AZD4573 (15 mg/kg; IP; once weekly), Copanlisib (15 mg/kg; IP; twice weekly), or a combination of both. Combo treatment restricted tumor growth and prolonged survival to a greater extent than either drug alone. Conclusions CDK9i with AZD4573 downregulated numerous oncoproteins. However, a subset of genes including MYC and PIM3 recovered transcription. PI3K/AKT pathway was implicated in resistance to CDK9i in CRISPR library screens. Concurrent targeting of pro-survival pathways (e.g., PIM, PI3K) partially reversed resistance to CDK9i. Citation Format: Elana Thieme, Duanchen Sun, Nur Bruss, Geeta Sharma, Tingting Liu, Daniel Coleman, Tamilla Nechiporuk, Daniel Bottomly, Shannon McWeeney, Patrick Pirrotte, Zheng Xia, Alexey Danilov. Strategies to circumvent resistance to cyclin-dependent kinase-9 inhibition (CDK9i) in non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A06.