Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia

JCI Insight · 2024-04-22 · 6 citations

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

NOD2 activation enhances macrophage Fcγ receptor function and may increase the efficacy of antibody therapy

Frontiers in Immunology · 2024-06-11 · 5 citations

Introduction: Therapeutic antibodies have become a major strategy to treat oncologic diseases. For chronic lymphocytic leukemia, antibodies against CD20 are used to target and elicit cytotoxic responses against malignant B cells. However, efficacy is often compromised due to a suppressive microenvironment that interferes with cellular immune responses. To overcome this suppression, agonists of pattern recognition receptors have been studied which promote direct cytotoxicity or elicit anti-tumoral immune responses. NOD2 is an intracellular pattern recognition receptor that participates in the detection of peptidoglycan, a key component of bacterial cell walls. This detection then mediates the activation of multiple signaling pathways in myeloid cells. Although several NOD2 agonists are being used worldwide, the potential benefit of these agents in the context of antibody therapy has not been explored. Methods: Primary cells from healthy-donor volunteers (PBMCs, monocytes) or CLL patients (monocytes) were treated with versus without the NOD2 agonist L18-MDP, then antibody-mediated responses were assessed. In vivo, the Eµ-TCL1 mouse model of CLL was used to test the effects of L18-MDP treatment alone and in combination with anti-CD20 antibody. Results: Treatment of peripheral blood mononuclear cells with L18-MDP led to activation of monocytes from both healthy donors and CLL patients. In addition, there was an upregulation of activating FcγR in monocytes and a subsequent increase in antibody-mediated phagocytosis. This effect required the NF-κB and p38 signaling pathways. Treatment with L18-MDP plus anti-CD20 antibody in the Eµ-TCL model of CLL led to a significant reduction of CLL load, as well as to phenotypic changes in splenic monocytes and macrophages. Conclusions: Taken together, these results suggest that NOD2 agonists help overturn the suppression of myeloid cells, and may improve the efficacy of antibody therapy for CLL.

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Blood · 2023-11-02 · 1 citations

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Active Hexose-Correlated Compound Shows Direct and Indirect Effects against Chronic Lymphocytic Leukemia

Nutrients · 2023-12-18

Chronic lymphocytic leukemia (CLL) is a disease characterized by the accumulation of mature CD19+CD5+CD23+ B cells in the bloodstream and in lymphoid organs. It usually affects people over 70 years of age, which limits the options for treatments. The disease is typically well-managed, but to date is still incurable. Hence, the need for novel therapeutic strategies remains. Nurse-like cells (NLCs) are major components of the microenvironment for CLL, supporting tumor cell survival, proliferation, and even drug resistance. They are of myeloid lineage, guided toward differentiating into their tumor-supportive role by the CLL cells themselves. As such, they are analogous to tumor-associated macrophages and represent a major therapeutic target. Previously, it was found that a mushroom extract, Active Hexose-Correlated Compound (AHCC), promoted the death of acute myeloid leukemia cells while preserving normal monocytes. Given these findings, it was asked whether AHCC might have a similar effect on the abnormally differentiated myeloid-lineage NLCs in CLL. CLL-patient PBMCs were treated with AHCC, and it was found that AHCC treatment showed a direct toxic effect against isolated CLL cells. In addition, it significantly reduced the number of tumor-supportive NLCs and altered their phenotype. The effects of AHCC were then tested in the Eµ-TCL1 mouse model of CLL and the MllPTD/WT Flt3ITD/WT model of AML. Results showed that AHCC not only reduced tumor load and increased survival in the CLL and AML models, but it also enhanced antitumor antibody treatment in the CLL model. These results suggest that AHCC has direct and indirect effects against CLL and that it may be of benefit when combined with existing treatments.

Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer

Cells · 2023-05-27 · 8 citations

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. METHODS: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. RESULTS: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. CONCLUSIONS: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.

Inhibition of BET Proteins Regulates Fcγ Receptor Function and Reduces Inflammation in Rheumatoid Arthritis

International Journal of Molecular Sciences · 2023-04-21 · 13 citations

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia

The Journal of Immunology · 2022 · 8 citations

• Cancer research
• Biology
• Immunology

cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

Abstract 1060: Introducing a novel DHODH inhibitor with superior <i>in vivo</i> activity as monotherapy or in novel combination regimen with immunotherapy for hematological malignancies

Cancer Research · 2022-06-15

Abstract Acute myeloid leukemia (AML) is characterized by the uncontrolled expansion of un-differentiated hematopoietic progenitor myeloblasts. AML treatment is very challenging owing to its complex heterogeneity resulting in a dismal 5-year overall survival rate particularly in elderly patients unfit for standard induction chemotherapy. The expansion of AML requires the availability of sufficient nucleotides supporting the anabolic processes required for AML growth thus, targeting nucleotide biosynthesis can halt AML progression. Indeed, targeting dihydroorotate dehydrogenase (DHODH), a critical rate-limiting step in the de novo pyrimidine synthesis pathway not only induced cytotoxicity but has been shown to promote blast differentiation in a HOXA9/MEIS1 over-expressing model. We sought to develop a DHODH inhibitor that had superior properties to those reported for AML therapy. Compound 41 (cmpd 41) demonstrates sub-nanomolar 50% inhibitory concentration for DHODH biochemical activity and potent in vitro activity across several AML cell lines and primary AML cells independent of mutational subtype, including mutated TP53. Cmpd 41 also demonstrated superior in vivo anti-leukemic activity in multiple AML xenograft models as monotherapy and demonstrated synergy with a hypomethylating agent, decitabine in TP53 mutated AML. Given the heterogeneity of AML and frequent emergence of resistant clones, we aimed to investigate ways to enhance response to DHODH inhibitors through combination. After in vitro treatment of AML cell lines and primary patient samples with DHODH inhibitors, we observed an increase in CD38 surface expression suggesting synergy with CD38 targeting monoclonal antibody (mAb) immunotherapies. Indeed, we are the first to report synergy between DHODH inhibitors and anti-CD38 mAb in AML which emphasizes the synergy between this promising novel class of agents with immunotherapies via recruiting innate immunity. Consequently, given the relevance of CD38 mAb therapy to multiple myeloma (MM), we extended these studies to MM and remarkably found that cmpd 41 was highly efficacious as a monotherapy and in combination with CD38 mAb, resulted in complete tumor regression in a subcutaneous MM xenograft model. In summary, we introduce a best in class DHODH inhibitor with a data-driven combination strategy for both AML and MM. Our studies suggest a highly synergistic combination strategy involving immunotherapy for AML and other hematologic malignancies. Citation Format: Ola A. Elgamal, Sandip Vibhute, Sydney Fobare, Abeera Mehmood, Mariah L. Johnson, Jean Truxall, Emily Stahl, Bridget Carmichael, Shelley J. Orwick, Ramasamy Santhanam, Kasey Hill, Susheela Tridandapani, Christopher C. Coss, Alice S. Mims, Karilyn T. Larkin, Mitch A. Phelps, Sharyn D. Baker, Alex Sparreboom, Thomas E. Goodwin, Gerard Hilinski, Chad E. Bennett, Erin Hertlein, John C. Byrd. Introducing a novel DHODH inhibitor with superior in vivo activity as monotherapy or in novel combination regimen with immunotherapy for hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1060.

RBIO-01. ACTIVATING AN ION CHANNEL PROMOTES RADIATION-SENSITIZING IMMUNE-DRIVEN ANTI-TUMOR ACTIVITY

Neuro-Oncology · 2022-11-01

Abstract Standard-of-care for melanoma brain metastases is moving to stereotactic radiosurgery (SRS) with immunotherapy. Although this combined approach shows improved patient survival, the responses are not durable. SRS can also cause adverse reactions such as radiation necrosis, which can result in significant morbidity and even mortality. Lowering SRS dose without negatively impacting its effectiveness would therefore be highly beneficial clinically. Our published data suggests that when treating syngeneic mouse model B16F10 melanoma tumors, the radiation dose can be reduced and its effectiveness can be improved, by activating intrinsic melanoma GABAA receptor (GABAAR) activity using a novel agent, AMLAL-101. This agent acts to enhance GABAAR mediated chloride transport in melanoma cells, which in turn triggers mitochondrial dysregulation associated with tumor regression in mice. While AMLAL-101 alone reduces tumor growth, combining it with radiation results in near complete regression of the irradiated tumor, as well as shrinking of tumor not in the radiation field due to an immune response, i.e., a potent abscopal effect. AMLAL-101 may also function to regulate immune cell infiltration into the tumor microenvironment. Analysis of AMLAL-101 treated tumors show increased infiltration into the tumor microenvironment of: (1) polyfunctional (TNFα/IFN-γ) CD8+ T cells; and (2) tumor-associated macrophages (TAMs) of the tumor inhibitory M1-phenotype, possibly due to IFN-γ stimulation. We posit that activating GABAAR with AMLAL-101 promotes both a ‘direct’ anti-tumor response and an ‘indirect’, immune-driven anti-tumor activity in metastatic melanoma tumors. AMLAL-101 may address an urgent unmet need for a non-toxic therapeutic that can potentiate radiation and mitigate its toxicity profile to positively impact clinical outcomes of patients with brain metastatic disease treated with radiotherapy.

Hosu-53, a Novel Potent Dihydroorotate Dehydrogenase Inhibitor Accentuates CD47 Blockade Therapy Resulting in Long Term Survival in Acute Myeloid Leukemia

Blood · 2022-11-15

Acute myeloid leukemia (AML) is a complex disease characterized by leukemia stem cells in a state of differentiation arrest leading to suboptimal innate immune function and immunosuppression. The effectiveness of allogeneic hematopoietic stem cell transplantation, NK cell directed therapies, and recently CD47 blockade highlights the importance of immune surveillance as potentially curative therapies for AML. Dihydroorotate dehydrogenase inhibitors (DHODHi) have been reported to have both cytotoxic and differentiation properties which propelled their clinical development for AML. We developed HOSU-53, a novel DHODHi which is described in detail in another abstract at this meeting. HOSU-53 has (i) nanomolar potency against human DHODH biochemical activity, (ii) in vitro anti-proliferative activity toward AML with a potent tumor DNA damage response, (iii) favorable oral bioavailability, (iv) a monitorable on-target pharmacodynamic biomarker predictive of efficacy and toxicity, and (v) superior in vivo survival outcome in disseminated AML cell line derived xenograft models. Prior anti-viral studies using DHODHi have shown that deprivation of pyrimidine synthesis and associated DNA damage response can enhance innate immune activation. Therefore, we hypothesized that HOSU-53 could enhance phagocytosis and antibody dependent cellular phagocytosis (ADCP) and thereby synergize with myeloid immune checkpoint blockade therapies. Herein, we report compelling preclinical data demonstrating curative potential in AML xenograft models. Treating AML cells with HOSU-53 resulted in restored ability to phagocytose E.coli suggesting enhanced innate immunity. Given our previous reports that DHODHi could induce cell surface markers such as CD38, we next validated that HOSU-53 can increase expression of calreticulin (CALR) and CD47. In vitro treatment with HOSU-53 demonstrated increased tumor surface expression of the pro-phagocytic signal, CALR and a dose dependent increase in CD47 expression. We performed ADCP assays using murine bone marrow derived macrophages and observed enhanced phagocytosis when AML cells were pretreated with HOSU-53, prompting us to conduct in vivo validation using the MOLM-13 disseminated model. Mice were enrolled four days post-engraftment to receive single agent anti-CD47 monoclonal antibody at 0.5mg daily for 21 days and either daily 4 or 10 mg/kg HOSU-53, based on our prior dose optimization studies. We also investigated the combination of CD47 antibody with each dose level of HOSU-53. All monotherapy cohorts resulted in significant prolonged survival, but still eventually succumb to AML disease. In contrast, the combination cohorts resulted in disease-free survival as determined by the absence of measurable human CD45+ cells in spleen or bone marrow cells harvested from mice at the end of treatment on day 80, or on day 106 after remaining on study for 25 days without continued treatment. The curative potential in this model prompted us to repeat our study in a model with higher tumor burden. In this follow-up study, mice were enrolled ten days post engraftment to receive daily HOSU-53 (4 or 10 mg/kg) or B6H12 anti-CD47 (0.5mg daily or every three days for 21 days) or combinations of these treatments. We observed that anti-CD47 monotherapy was ineffective with this more established tumor model, but HOSU-53 monotherapy was comparable to our previous study and both 10 mg/kg combination cohorts significantly prolonged survival (Fig. 1, ongoing study). Further mechanistic studies of HOSU-53-mediated synergy of CD47 therapy are ongoing. Herein, using our DHODHi HOSU-53, we describe that pyrimidine depletion can enhance innate immunity to potentiate CD47 blockade therapy, representing a novel treatment approach. We show HOSU-53 increased tumor innate immune function, CALR expression and subsequently increased in vitro phagocytosis with CD47 blockade. Furthermore, in vivo combination of HOSU-53 with anti-CD47 antibody promoted dramatic synergy exhibiting a potentially curative approach in an otherwise very aggressive tumor model. HOSU-53 is currently in the IND-enabling studies phase with plans for filing an IND application in 2023 for phase 1 clinical trials in AML and MM. This data and our previously reported combination of HOSU-53 with anti-CD38 antibodies warrants the clinical translation of DHODHi in combination with immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal