Page 98 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
and clonal expansion of immature myeloid cells, leading to ineffective hematopoiesis. In the past, standard of care therapy has been limited to cytotoxic chemotherapy such as cytarabine and anthracycline. However, since 2017 several targeted therapies were approved by the Food and Drug Administration as viable therapeutic options. The BCL-2 inhibitor VEN has demonstrated particular success, which has emerged as a critical component to the standard of care in multiple lymphoid and myeloid neoplasms. As a single agent, VEN demonstrates mod- est activity in relapsed/refractory AML,11 yet when used in combination with a DNA methyltransferase inhibitor (DNMTi)12 or low-dose cytosine arabinoside (LDAC) led to remission in 73% of newly diagnosed elderly patients.13-15 Unfortunately, despite these encouraging response rates, most patients treated with VEN will eventually relapse. Previous studies have suggested mechanisms of resis- tance for both primary and acquired BCL-2 resistance include increased levels of MCL-1 as well as mutations in TP53.12,16,17 Increasing and prolonging VEN efficacy in hematologic malignancies remains a priority.
We first sought to test CD47 inhibition in the context of an AML VEN-based regimen to determine if efficacy could be increased. We added ALX90 to a standard of care VEN/azacitidine (AZA) regimen in multiple AML in vivo models. In cell line-derived xenografts (CDX) mod- els where medullary tumor burden is low (<50% cell of bone marrow [BM] cell volume at moribund) and native macrophages are present at endpoint found that CD47 inhibition led to a significant decrease in tumor bur- den as a monotherapy, and augments standard of care therapy with VEN/AZA. However, in patient-derived xe- nograft (PDX) models with high tumor burden and low native medullary macrophage constituency, CD47i failed to decrease medullary tumor burden as a monotherapy, despite almost complete clearance of tumor cells with- in the peripheral blood and spleen. Within the paucity of macrophages present in the marrow of PDX models, almost all were polarized toward M2. This implied that the small population of tumor-supportive M2 macro- phages, and lack of M1 tumor-suppressive macrophages limited the potential of CD47 inhibition. Thus, in an at- tempt to increase medullary macrophage function in mice, we co-treated with poly(I:C), a toll-like receptor 3 (TLR3) agonist which mimics viral double stranded RNA and is known to stimulate macrophage differentiation and polarization to M1 macrophages.18-20 As expected, in naïve NSGS mice, poly(I:C) transiently increased M1 macrophage populations in bone marrow, splenic, and blood compartments. Surprisingly, when poly(I:C) was combined with a VEN/AZA + ALX90 in PDX models it led not only to significant decreases in tumor burden in all compartments, but allowed for return to normal murine hematopoiesis and healthy BM. Poly(I:C) combined with ALX90 likewise reduced patient-derived leukemia in the
BM of immunocompromised mice. These data suggest that in the setting of AML, the presence of active BM M1 macrophage populations is required to induce a mean- ingful response to CD47i. We demonstrate here that by increasing BM macrophage function in murine models of AML, a more effective immune microenvironment may be restored, thereby potentiating CD47i in AML.
Methods
Patient samples
Experiments were conducted on primary patient samples which were provided by the Vanderbilt-Ingram Cancer Center Hematopoietic Malignancies Repository and are in accordance with the tenets of the Declaration of Helsinki and approved by the Vanderbilt University Medical Center Institutional Review Board.
Cell lines
AML cell lines MV-4-11 and THP-1 were purchased from the American Type Culture Collection (Manassas, VA). The MOLM-13 cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Ger- many). ATCC and DSMZ cell lines are authenticated by short tandem repeat profiling and cytochrome C oxidase gene analysis. Cells were used for the experiments presented here within 10-30 passages from thawing. MV-4-11 cell line was grown in IMDM, and all other cell lines were cultured in RPMI and supplemented with 10-20% fetal bovine serum and 100 U/mL penicillin and 100 ug/mL streptomycin. Cells were kept at 37°C in a 5% CO2 incubator.
In vivo murine model
All animal studies were conducted in accordance with guidelines approved by the IACUC at Vanderbilt University Medical Center. Female NSGS [NOD/SCID/Tg(hSCF/hGM- CSF/hIL3)] mice, 6-8 weeks old were irradiated with 1 Gy microwave radiation. Twenty-four hours later, mice were injected with 1x106 MV-4-11, 3x103 MOLM-13 or 2x106 AML leukapheresis primary patient cells via tail vein injection. Leukapheresis sample description is included in the Online Supplementary Appendix. After establishing microchime- rism, 1-2 weeks post-transplant, mice were treated with either VEN and AZA (VEN 20 mg/kg 5 days [D] on 2 D off, AZA upfront Monday/Wedneday/Friday [M/W/F] 1.5 mg/kg), with and without ALX 90 (30 mg/kg M/W/F), the combina- tion, or vehicle. Poly(I:C) (Invivogen) was dosed at 15 mg/kg M/W/F every 2 weeks, with 1 week off between cycles, for up to 8 weeks. Peripheral blood draws were taken weekly to assess chimerism.
Flow cytometry
For flow cytometry of murine blood and tissues, red blood cells were lysed with EL Buffer on ice (Qiagen), with remain-
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