Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, ...and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (
) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in
mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in
mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.
Peripheral T-cell Lymphoma (pTCL) make up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor outcomes to standard ...of care therapy. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved. CDK9 regulates transcription elongation through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor, acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are frequently overexpressed in haematologic tumours. It has been previously shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression and dependencies of the anti-apoptotic BCL-2 family members is highly heterogeneous in pTCL; however MCL-1 is the most universally expressed BCL-2 family member. Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition to MCL-1, we have previously reported the importance of the anti-apoptotic protein BFL-1 in mediating cell survival in NHL, including a subset of pTCL (Boiko et al 2021). Consistent with these findings, AZD4573 treatment exhibited a range of activity across the panel of 18 human TCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, having EC 50 values < 100 nM, and 13 reached a max caspase-3/7 activation of greater than 40%. pTCL cell lines that responded to AZD4573 treatment including ALCL, NK-TCL, and PTCL-NOS and CTCL subtypes. Acute AZD4573 treatment of pTCL cell lines resulted in decreased p-SerRNApoll II and reduced levels of c-MYC and MCL-1 protein levels. To determine if MCL-1 or c-MYC was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 after long term KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that AZD4573 efficacy is mediated through MCL-1, in ALCL pTCL cell lines tested. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and assessed for viability after 24-72 hours using RealTime-Glo after 18hr exposure to CHOP and AZD4573 added for the last 6hrs. Adding AZD4573 treatment to CHOP schedule in vitro increased the combination benefit in pTCL ALCL cell lines that show MCL-1 dependency, but not in CTCL cell line which show a BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels at 24-48 hours, but not MCL-1 suggesting that combination benefit may be driven through decreased c-MYC levels, as KO of c-MYC resulted is loss of cell viability in these models. These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.
Burkitt Lymphoma (BL) accounts for almost two thirds of all B-cell non-Hodgkin lymphoma cases in children and adolescents. It may affect the jaw, bones of the face, bowel, kidney, ovaries and in some ...cases spread to the central nervous system. Nearly all types of BL are characterized by c-MYC gene translocation of chromosome 8 with immunoglobulin genes at chromosome 2, 14 or 22 resulting in high levels of the transcription factor. Recently, MCL-1 was shown to be overexpressed in BL and is a critical survival factor in pre-clinical BL models using genetic and chemical approaches. CDK9, a serine/threonine kinase, regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). AZD4573 is a highly potent and selective CDK9 inhibitor that results in downregulation of short-lived transcripts and labile proteins such as c-MYC and key survival proteins including MCL-1 and BFL-1, resulting in apoptosis in several hematological malignancies (Cidado et al. 2020 & Boiko et al. 2021) We selected several preclinical BL models to assess the rapid apoptogenic potential of AZD4573 in vitro and in vivo. To measure apoptosis, we evaluated cleaved caspase-3 (CC3) induction following acute treatment (6h) using Caspase-Glo 3/7. Using this assay, we found that 3 BL models were sensitive to CDK9 inhibition (EC50 < 100nM; max. CC3 > 50%) and 2 were resistant (EC50 > 100nM; max. CC3 < 50%). Treatment with AZD4573 in these sensitive BL cell line models decreased pSer2-RNAPII, and downregulation of MCL-1 and c-MYC within 6 hours. In AZD4573 resistant lines we observed relatively higher levels of anti-apoptotic proteins BCL2 and BCL-xL, compared to the sensitive cell lines. These increased levels potentially lead to the decreased sensitivity to AZD4573, which does not reduce levels of BCL-2 and BCL-xL due to their longer half-life. Using CRISPR genetic knockouts we demonstrate that BL cell lines are dependent on c-MYC and MCL-1 and that loss of these protein decreases caspase induction after AZD4573 treatment, highlighting the importance of these proteins in driving the efficacy of AZD4573. In vivo AZD4573 dosed on a once weekly schedule resulted in tumor growth inhibition in aggressive BL cell line xenografts Namalwa and Ramos (40-60%) and decreased pSer2-RNAPII, MCL-1 and c-MYC and increased CC3 8 hours after treatment. AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.
Acute myelogenous leukemia (AML) comprises a highly polyclonal and heterogeneous disease containing leukemic stem cells, progenitors, and differentiated blasts. Despite advances in treatment, the ...long-term relapse-free survival for AML remains poor. Due to complex mutational profiles and highly divergent subtypes in AML, the use of physiologically relevant, patient-derived models is needed. Traditional cell line models of AML almost exclusively represent differentiated blasts, are homogenous in clonality, and often do not encompass major genetic subtypes. To address these gaps, we have established a culture system that supports the growth of higher-order AML samples ex vivo. By optimizing culture conditions, we successfully cultivated 70% (10/14) of AML disseminated PDX models ex vivo and demonstrated they continue to proliferate after long term cultures. We established a 16-color flow panel to characterize their cell maturity state and show they maintain cell surface heterogeneity. Importantly, these models have not ‘drifted’ transcriptomically after multiple passages. Our platform includes 2 NPM1c mutant and 3 IDH1/2 mutant ex vivo lines, in addition to other subtypes underrepresented in traditional culture methods. Furthermore, we treated AML PDXs in vivo with a clinically-relevant dose of venetoclax + azacytidine (Ven/AZA) for 2 cycles (14 days) and then evaluated post-VEN/AZA cells in our ex vivo assay. Growing these models ex vivo, we show using our flow panel that Ven/AZA treatment shifts the differentiation status from a stem-like state to a more mature-like cell state which is consistent with literature evidence of treatment resistance. Additionally, a second PDX model treated with three cycles of Ven/AZA in vivo and harvested in ex vivo culture conditions maintained both Ven and AZA resistance. Interestingly, compared with the vehicle treatment, Ven/AZA treatment caused resistance to other cell death agents including selective MCL1 and dual Bcl2/xL inhibitors. Lastly, we performed a 7-day cell compound panel screen using common standard of care agents for the treatment of AML. Many of the targeted agent activity correlated with expected genetic subtypes, such as FLT3 inhibitors in FLT3-ITD mutants and venetoclax in WT TP53 models. This established ex vivo platform provides physiologically relevant models to explore novel target biology, better understand compound mechanism of action (MoA), and generate drug resistant models through in vivo driven clinically relevant dose scheduling using standard of care agents.
Abstract
Relapsed/refractory DLBCL is an aggressive B-cell malignancy with limited treatment options. BTK inhibitors have demonstrated preclinical and clinical activity in DLBCL of the activated ...B-cell (ABC) subset, but responses are limited and not durable. Therefore, an acalabrutinib (BTK inhibitor) combination screen was conducted in a panel of 9 DLBCL cell lines to identify synergistic and active combinations. The in vitro combination of acalabrutinib plus capivasertib (AKT inhibitor) demonstrated significant combination benefit in the ABC-DLBCL cell lines TMD8 (Loewe synergy score 9.3) and OCI-LY10 (Loewe synergy score 3.2). Capivasertib and acalabrutinib monotherapy gave 0% and 85% tumor growth inhibition (TGI) in TMD8 and 5% and 79% in OCI-LY10 tumour xenograft models respectively. The combination gave tumour regressions of 99% in TMD8 and 72% in OCI-LY10. To explore a potential mechanism of action, RNAseq analysis was performed on the TMD8 cell line and TMD8 xenograft tumors treated with the monotherapies and combination. 24-hour monotherapy treatment of TMD8 cells with acalabrutinib altered the expression of 7390 genes while capivasertib altered 398 genes (absolute fold change ≥ 1.25 and adjusted p-value < 0.05), with enrichment for genes regulated by NFkB signalling detected. Pathway analysis following combination treatment revealed significant decrease in expression of the G2M checkpoint pathway genes compared to acalabrutinib alone (Adjusted p-value = 0.007) as well as decreased expression of CDK1, AURKA and MYC, and significant shifts in the TNFa signalling via NFkB pathway (Adjusted p-value = 0.02). The NFkB signalling node was examined in more detail in TMD8 tumor samples treated with the combination. Lower expression of NFKBIA, NFKBID, EGR2 and BCL2A1 was evident in the combination group, compared to control tumors (p<0.01 for all). Interestingly, the combination strongly increased the expression of MS4A2 (CD20) compared to control (Adjusted p-value = 0.002), capivasertib monotherapy (Adjusted p-value = 0.004) and acalabrutinib monotherapy (Adjusted p-value = 0.02). Therefore, we tested the addition of Rituxan to acalabrutinib + capivasertib combination in the TMD8 xenograft model. The triple combination produced durable complete regressions in 5/5 mice after cessation of treatment whereas tumors regrew after cessation of treatment of the acalabrutinib + capivasertib doublet. These data suggest that the combination of BTK and AKT inhibition may enhance anti-tumor activity in ABC DLBCL, with the addition of anti-CD20 giving more durable tumour control.
Citation Format: Kathleen Burke, Justine Roderick-Richardson, Natasha Narang, Brandon Willis, Hannah Dry, Lillian Castriotta, Alan Rosen, Jay Mettetal, Simon Barry, Andrew Bloecher. Combination activity of acalabrutinib and capivasertib in diffuse large B-cell lymphoma 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 1024.
Abstract Background: Acute myeloid leukemia (AML) is a prevalent adult leukemia. In the US, around 20,380 individuals were diagnosed with AML in 2023. Despite standard therapies, relapse and ...resistance persist. Cyclin-dependent kinase 9 (CDK9) has been recognized an actionable therapeutic target in AML. The potent inhibitor, AZD4573, induces durable apoptosis and suppresses tumor growth. Our study reveals that combining AZD4573 with standard-of-care (SoC) treatments deepens cell death responses in multiple AML cell models. We also used proteomics tools to understand the mechanisms underlying the vulnerability and resistance in SoC and CDK9 combinations. Methods: In the drug combination study, three SoC agents (cytarabine, decitabine, and venetoclax) were evaluated in combination using a 6 × 6 dose-response matrix in four AML cell models (MOLM13, MOLM16, MV411, and OCI-AML2). The HSA synergy score and Emax drug activity were used to assess the benefits of the drug combinations. In the proteomics evaluation study, MOLM13 cells treated for 7 days under different conditions - (1) vehicle control; (2) cytarabine alone; (3) decitabine alone; (4) venetoclax alone; (5) AZD4573 alone; (6) AZD4573+cytarabine; (7) AZD4573+decitabine; (8) AZD4573+venetoclax - were collected for proteomics analysis using LC-MS/MS. Differential expression and missing pattern analyses were used to identify dysregulated proteins. Functional enrichment analysis was employed to identify dysregulated biological processes and pathways in response to different treatments. Results: Our drug combination screens demonstrated the efficacy of AZD4573 in combination with cytarabine, decitabine, and venetoclax in 3 of 4 AML cell models. In our proteomic analysis, we took two approaches. First, we compared the proteomes of AML cells treated with SoC agents, identifying shared downregulated pathways such as serine biosynthesis, notably pointing to the rate-limiting enzyme PHGDH as a potential therapeutic target in AML. Cytarabine and decitabine induced an inflammatory response, while venetoclax increased the expression of cell division regulators like Aurora kinase B (AURKB). Interestingly, targeting AURKB with AZD2811 has been shown to overcome venetoclax resistance in AML. Second, we compared proteome changes induced by various AZD4573 combinations. We identified IRF2BP2 as a shared vulnerable protein across the three AZD4573 combinations. POUF2F2 expression was significantly induced by cytarabine but suppressed when combined with AZD4573. Given that POUF2F2 activates the PDK1-Akt pathway, our findings suggest that AZD4573-induced POUF2F2 downregulation suppresses the PDK1-AKT pathway, inhibiting tumor progression. Conclusions: Our proteomics analysis identifies vulnerable and resistant pathways and proteins, shedding light on the mechanism of action and revealing potential new targets for AML. Citation Format: Lori Chan, Zuleyha Ozen, Anthony Iannetta, Eric Miele, Funda Kar, Andrew Jarnuczak, Geoff Nelson, Aleksandra Markovets, Justine Roderick-Richardson, Jelena Urosevic, Lisa Drew, Jerome Mettetal. Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7089.
