MS-553 and venetoclax display synergy.

Abstract Chronic graft-versus-host disease (cGVHD) significantly contributes to late mortality after allogeneic stem cell transplantation, with bronchiolitis obliterans syndrome (BOS) being a particularly lethal and treatment-resistant complication despite available therapies. Bromodomain and extraterminal (BET) proteins are epigenetic readers driving inflammatory transcriptional programs across multiple cell types. We hypothesized that BET inhibition would suppress inflammatory T and B cells and decrease macrophage polarization to a profibrotic phenotype, alleviating disease. In an established BOS cGVHD model, BET inhibition reduced germinal center (GC) formation and responses through a reduction of the CXCL13:CXCR5 axis and inflammatory T follicular helper/GC B cells in the spleen, along with a reduction in plasma cell infiltration within the lung. Mice with cGVHD had elevated pathogenic immunoglobulin G1 (IgG1) and IgM levels, both in circulation and deposited on lung tissue, which were attenuated under BET inhibition. Single-cell RNA-sequencing analysis revealed distinct cell states in the BOS lung vs control. In cGVHD mice, gene set enrichment analysis revealed the upregulation of profibrotic Arginase1 and Tgfb1 expression in alveolar macrophages (AM) and interstitial macrophages (IM), which was significantly reduced with BET inhibition. Furthermore, BET inhibition targeted lung-infiltrating M2 macrophages through the selective depletion of CD206+FcγR+ IM and AM, ultimately resulting in reduced collagen deposition and improved lung function. Our findings reveal a previously unrecognized mechanistic axis of BET regulation during cGVHD fibrosis and highlight BET inhibition as a promising therapeutic strategy.

MS-553 inhibits downstream BCR and Wnt/β-catenin signaling in BTKi resistant CLL.

Effect of MS-553 on Wnt reporter cell line.

Effect of MS-553 on PKCβ and PKCε in CLL.

Clinical characteristics of BTKi resistant CLL patient samples

Effect of MS-553 on BCR Signaling.

Gating strategy for disease progression and effects on T- and NK-cell populations.

Characteristics of CLL patients with dual-refractory disease

PKCβ is accessible and upregulated in dual-refractory CLL.

Treatment history of patients used for scRNA-seq and scATAC.

Effect of MS-553 towards co-culture systems.

Inhibition of the tyrosine kinase BTK is a major therapeutic strategy for treating B-cell malignancies, including chronic lymphocytic leukemia (CLL). However, resistance can emerge when tumor cells acquire mutations that abrogate drug binding or when BTK activates B-cell receptor (BCR) signaling by mechanisms independent of its kinase activity. In this study, we identified upregulation of PKCβ in samples from patients with CLL resistant to BTK inhibitors (BTKi) and characterized the PKCβ inhibitor MS-553. MS-553 reduced BCR and Wnt/β-catenin signaling, overcame stromal cell mediated protection, and synergized with venetoclax in CLL samples. MS-553 also retained the cytotoxicity and inhibition of both BCR and Wnt/β-catenin signaling in models (cell lines and primary samples) of covalent BTKi-resistant (C481S BTK) and noncovalent BTKi-resistant (T474I or L528W BTK) CLL. Furthermore, MS-553 delayed disease progression and prolonged survival in the Eμ-MTCP1 murine model of CLL. Collectively our results demonstrate that selective inhibition of PKCβ has the potential to overcome BTKi-resistant CLL. SIGNIFICANCE:This study identified and characterized PKCβ as a therapeutic target in CLL, including CLL resistant to BTKis. Inhibition of PKCβ suppressed both BCR and Wnt/β-catenin signaling, delayed disease progression in vivo, overcame BTKi resistance mechanisms, and enhanced response to BCL-2 inhibition. See related commentary by Jebaraj and Stilgenbauer, p. 21.

Gating strategy for Cytochrome c release and Annexin V/PI.

Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes, which limits broad applicability of targeted therapy. We discovered that the hematopoiesis-restricted tetraspanin CD37 is expressed on the majority of primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared with normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody-drug conjugate (ADC) Debio 1562 (alpha CD37DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that alpha CD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as alpha CD37DM1 is a feasible and promising therapeutic option for the treatment of AML.

This phase 1 study investigated the addition of gemtuzumab ozogamicin (GO) to intensive chemotherapy with cytarabine, daunorubicin, and midostaurin in 21 patients with newly diagnosed (ND) FMS-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML). Four dose levels of GO were evaluated. The use of GO was tolerable, with all dose-limiting toxicities similar to those seen in standard-of-care treatment. After induction, the median time to platelet recovery was 26 days, and the median time to absolute neutrophil count (ANC) recovery was 27 days. The maximum tolerated dose was cytarabine 100 mg/m2 on days 1 to 7, midostaurin 50 mg twice daily on days 8 to 21, daunorubicin 60 mg/m2 on days 1 to 3, and GO 3 mg/m2 on days 1 and 4. For the 18 patients who were evaluable for response after induction therapy, 16 patients (76%) achieved a composite complete response (complete remission [CR] + CR with incomplete hematologic recovery), and 2 (10%) had stable disease. Of the 14 patients who proceeded to consolidation, 5 discontinued the study for transplant, 1 for disease progression, and 1 for physician discretion. Seven patients completed consolidation therapy, all of whom achieved a CR. In total, 13 of the 21 patients (62%) received a hematopoietic stem cell transplant. Our results show that GO can safely be combined with intensive chemotherapy with midostaurin in ND, FLT3-mutated AML. This trial was registered at www.clinicaltrials.gov as #NCT03900949.

Mutations and deletions in TP53 are associated with adverse outcomes in patients with myeloid malignancies, and there is an urgent need for the development of improved therapies for TP53-mutant leukemias. Here, we identified mutations in TET2 as the most common co-occurring mutation in patients with TP53-mutant acute myeloid leukemia (AML). In mice, combined hematopoietic-specific deletion of TET2 and TP53 resulted in enhanced self-renewal compared with deletion of either gene alone. Tp53/Tet2 double-KO mice developed serially transplantable AML. Both mice and patients with AML with combined TET2/TP53 alterations upregulated innate immune signaling in malignant granulocyte-monocyte progenitors, which had leukemia-initiating capacity. A20 governs the leukemic maintenance by triggering aberrant noncanonical NF-κB signaling. Mice with Tp53/Tet2 loss had expansion of monocytic myeloid-derived suppressor cells (MDSCs), which impaired T cell proliferation and activation. Moreover, mice and patients with AML with combined TP53/TET2 alterations displayed increased expression of the TIGIT ligand, CD155, on malignant cells. TIGIT-blocking antibodies augmented NK cell-mediated killing of Tp53/Tet2 double-mutant AML cells, reduced leukemic burden, and prolonged survival in Tp53/Tet2 double-KO mice. These findings describe a leukemia-promoting link between TET2 and TP53 mutations and highlight therapeutic strategies to overcome the immunosuppressive bone marrow environment in this adverse subtype of AML.

Venetoclax creates ongoing challenges when combined with posaconazole due to a known drug-drug interaction. Herein, we investigated the safety between venetoclax 100mg and 70mg when administered with posaconazole in acute myeloid leukemia in this single-center, retrospective comparative analysis. Primary safety endpoints were incidence/duration of cytopenias and incidence of tumor lysis syndrome during the first treatment cycle. A total of 113 patients received venetoclax 100mg while 32 patients received 70mg. Comparing venetoclax 100mg vs 70mg, no statistically significant differences were seen in grade 3 neutropenia (89.4% vs 84.4%, p=0.53), grade 4 neutropenia (88.5% vs 87.5%, p=1.0), median duration in days of grade 4 neutropenia (23 [range 1–105] vs 28 [range 1-81], p=0.35), grade 3/4 anemia (88.5% vs 84.4%, p=0.55), grade 3/4 thrombocytopenia (81.4% vs 87.5%, p=0.42), or tumor lysis syndrome (2.7% vs 6.3%, p=0.30). In adult patients with acute myeloid leukemia, a target dose of venetoclax 100mg with posaconazole may be a safe alternative. Further studies assessing dose optimization are warranted.

Introduction AML-MR is defined by cytogenetic and/or molecular abnormalities associated with myelodysplastic neoplasms or arising from a known history of myelodysplastic/ myeloproliferative neoplasms. T-AML is characterized by prior cytotoxic, radiation, or immunosuppressive therapy for an unrelated disease. Pts with AML-MR and t-AML have worse outcomes historically (Granfeldt Ostgard et al. J Clin Oncol 2015;33:3641). In the VIALE-A trial, only about 25% of pts had secondary AML (DiNardo et al. N Engl J Med 2020;383:617). In the VIALE-C trial, pts with secondary AML (38%) had worse survival than those with de novo AML (Wei et al. Blood 2020;135:2137). Both studies excluded pts with prior hypomethylating agent (HMA) exposure for prior MDS/MPN. Methods This study was a single-center, retrospective analysis of pts with AML-MR or t-AML, defined according to WHO 2022 classification (Khoury et al. Leukemia 2022;36:1703), who were treated with a venetoclax-based regimen from 10/2016 to 5/2023. Clinical, cytogenetic and molecular data were collected to establish diagnosis of AML-MR and t-AML. CRh, CRi, and CR were determined based on the 2022 ELN guidelines. Measurable residual disease (MRD) status measured by multiparameter flow cytometry was collected after cycles 2 and 3, with an MRD-positive threshold of ≥0.1%. Overall survival (OS) was calculated from time of venetoclax initiation. Early mortality was defined as death within 30 days after diagnosis. Kaplan-Meier method and the log-rank test were used to assess survival differences between AML-MR vs non-AML-MR and t-AML vs non-t-AML pts. Results Ninety-five pts who received venetoclax-based therapy were included. Median age was 67 years (range, 23-92), and 40 (42%) were female. Sixty-nine pts (73%) had AML-MR and 22 pts (23%) had t-AML. Fifteen pts had both AML-MR and t-AML. There was no significant difference in age, sex, or ECOG performance status between pts with AML-MR and those with non-AML-MR. Venetoclax first line was received by 30% of pts with AML-MR and 35% of those with non-AML-MR. Venetoclax was combined with HMA in 88% of pts with AML-MR and 96% of pts with non-AML-MR. The AML-MR group completed a median of 2 cycles (range, 1-14) and the non-AML-MR group completed a median of 4 cycles (range, 1-56) of venetoclax-based therapy. There were no significant differences in CRh, CRi, and CR rates between AML-MR vs non-AML-MR (composite CR/CRh/CRi 35% vs 46%). Among 25 pts with AML-MR who had MRD assessment at cycles 2 or 3, 12 (48%) were MRD-positive and 13 (52%) MRD-negative, whereas among 15 pts with non-AML-MR, 6 (40%) were MRD-positive and 9 (60%) MRD-negative. After excluding pts post-allogeneic transplant, there was no significant difference in the number of pts who proceeded to transplant [11/18 (61%) for AML-MR v. 54/70 (77%) for non-AML-MR, p = .23]. There was no significant difference in OS (median, 35.1 vs 41.3 months, p=0.85). Three pts with AML-MR and 0 with non-AML-MR suffered early mortality (p=0.56). There was no significant difference in age, sex or ECOG performance status between pts with t-AML and those with non-t-AML. Fifty percent of pts with t-AML and 33% with non-t-AML received venetoclax first line. Venetoclax with HMA was administered to 91% of pts with t-AML and 90% with non-t-AML. Pts with t-AML completed a median of 2 cycles (range, 1-13) and those with non-t-AML completed a median of 3 cycles (range, 1-56) of venetoclax-based therapy. There was no significant difference in CRh, CRi, and CR rate between t-AML vs non-t-AML (composite CR/CRh/CRi 27% vs 41%). Of the pts with t-AML who had MRD assessment at cycles 2 or 3, 5 (56%) were MRD-positive and 4 (44%) MRD-negative. Among pts with non-t-AML, 13 (42%) were MRD-positive and 18 (58%) were MRD-negative. After excluding pts post-allogeneic transplant, there was no significant difference in the number of pts who proceeded to transplant [4/22 (18%) with t-AML v. 14/66 (21%) with non-t-AML, p=1.00]. There was no significant difference in OS (median, 41.3 vs 35.1 months, p=0.74). One pt with t-AML and 2 pts with non-t-AML suffered early mortality. Conclusions In our real-world cohort, pts with AML-MR and those with t-AML treated with venetoclax had no significant differences in outcomes (CR, OS, and early death) compared with pts with non-AML-MR and non-t-AML, respectively. This data supports further investigation into the treatment of AML-MR and t-AML with venetoclax-based regimens.

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with therapies targeting Bruton's tyrosine kinase (BTK) and BCL-2, but are limited due to the development of resistance. Dual targeting of BTK and BCL-2 has been efficacious in the clinic, with many patients achieving uMRD and prolonged remission off therapy. We have previously demonstrated the pre-clinical efficacy of LP-168 (Rocbrutinib), a novel selective 4th generation BTKi with an active warhead capable of covalent interaction with WT and T474I BTK and non-covalent binding when a BTK C481 mutation is present (Gordon et al. 2023). This allows for continued BTK inhibition despite development of common resistance mutations. To explore potential combination strategies with LP-168, we utilized a genome wide CRISPR/Cas9 knockout screen with validation of identified targets. Methods: Genome wide CRISPR/Cas9 knockout screening was performed in HG-3 CLL cells using the Brunello library with analysis via the MAGeCK pipeline. Cells were treated with IC20 (1 uM) of LP-168 for 3 days. Following exclusion of essential genes, negatively selected sgRNAs that were differentially expressed in the LP-168 group were used for analysis. Validation of hits was performed via pharmacological inhibition utilizing CellTiter-Glo, Annexin V/PI, and western blots using HG-3, OSU-CLL, TMD8 WT BTK, TMD8 C481S BTK, and TMD8 T474I BTK cells in addition to primary patient samples. Experiments using TMD8 cells were performed following CRISPR modification to insert either C481S or T474I BTK. Results: The CRISPR/Cas9 screen revealed 1875 genes with at least 3 out of the 4 sgRNAs depleted in both replicates of the LP-168 treated group vs control. KEGG/GO analysis demonstrated significant enrichment of regulation of mitochondrial membrane potential (p=0.00195) and reactive oxygen species pathways (p=0.00195). As BCL-2 interacts with both pathways, and was amongst the top-ranking genes depleted, we chose to target it with either venetoclax (BCL-2i) or with LP-118, a dual BCL-2/-xL inhibitor currently under investigation for R/R hematological malignancies. Pharmacologic inhibition of BTK + BCL-2, with either venetoclax (ven) or LP-118, showed synergistic reductions in proliferation of HG-3 (p<0.05, n=3; p<0.05, n=3, respectively), OSU-CLL (p<0.001, n=3, p<0.0001, n=3, respectively), and TMD8 WT BTK (p<0.05, n=3; p<0.01, n=3, respectively) cells at physiologically achievable concentrations (10 nM - 1 uM LP-168 and 10 pM - 10 nM ven or LP-118). LP-168 + ven or LP-118 induced significant cytotoxic effects in OSU-CLL (11%, p=0.001, n=3; 21%, p<0.00001, n=3, respectively) and TMD8 WT BTK (36.4%, p<0.00001, n=3; 66.7%, p<0.00001, n=3, respectively) cells following 72-hour drugging. Next, we utilized primary CLL B cells from treatment-naïve patients and found after both a 24- or 48-hour exposure, the combination of 1 uM LP-168 + 4 nM LP-118 (88%, p<0.00001, n=8) or 1 uM LP-168 + 4 nM ven (73%, p<0.00001, n=8) was able to induce cytotoxicity. Following a 2-hour drugging, LP-168 inhibited both BCR and AKT/mTOR signaling in primary CLL B-cells shown by reduced phosphorylation of BTK, ERK, AKT, and GSK3β, with both combination treatments inducing further inhibition of these targets (98%, p<0.0001, n=4; 49%, p<0.05, n=4; 37%, p<0.05, n=4; 46%, p<0.01, n=4, respectively). Further, we observed retained synergistic effect with both covalent (C481S; p<0.01, n=3) and non-covalent (T474I; p<0.001, n=3) BTKi resistance mutations at physiologically relevant concentrations. Finally, to determine if these combinations retain efficacy in both BTKi and venetoclax resistant settings of CLL, we utilized patient samples who were resistant to both BTKi and ven and treated them with combination of LP-168 + ven and found both combinations retained cytotoxic effect following 24- or 48-hour exposure. Conclusions : These data show combined use of LP-168 with pharmacological inhibitors targeting BCL-2 and BCL-xL display synergistic activity in CLL, even in the presence of mutations that mediate resistance to BTKi and BCL-2i. These data are consistent with clinical data showing dual targeting of BTK, with previous generation inhibitors, and BCL-2 has been effective in the clinic, with many patients achieving uMRD and prolonged remission off therapy and supports continued preclinical and future clinical investigation of LP-168 with inhibitors of BCL-2 and BCL-2/-xL.