Allison Banuelos

All Publications

• Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal. Proceedings of the National Academy of Sciences of the United States of America Banuelos, A., Baez, M., Zhang, A., Yılmaz, L., Kasberg, W., Volk, R., Georgeos, N., Koren-Sedova, E., Le, U., Burden, A. T., Marjon, K. D., Lippincott-Schwartz, J., Zaro, B. W., Weissman, I. L. 2025; 122 (21): e2426644122

  Abstract

  Calreticulin (CALR) is primarily an endoplasmic reticulum chaperone protein that also plays a key role in facilitating programmed cell removal (PrCR) by acting as an "eat-me" signal for macrophages, directing their recognition and engulfment of dying, diseased, or unwanted cells. Recent findings have demonstrated that macrophages can transfer their own CALR onto exposed asialoglycans on target cells, marking them for PrCR. Despite the critical role CALR plays in this process, the molecular mechanisms behind its secretion by macrophages and the formation of binding sites on target cells remain unclear. Our findings show that CALR undergoes C-terminal cleavage upon secretion, producing a truncated form that functions as the active eat-me signal detectable on target cells. We identify cathepsins as potential proteases involved in this cleavage process. Furthermore, we demonstrate that macrophages release neuraminidases, which modify the surface of target cells and facilitate CALR binding. These insights reveal a coordinated mechanism through which lipopolysaccharide (LPS)-activated macrophages regulate CALR cleavage and neuraminidase activity to mark target cells for PrCR. How they recognize the cells to be targeted remains unknown.

  View details for DOI 10.1073/pnas.2426644122

  View details for PubMedID 40397678

• Engineered CD47 protects T cells for enhanced antitumour immunity. Nature Yamada-Hunter, S. A., Theruvath, J., McIntosh, B. J., Freitas, K. A., Lin, F., Radosevich, M. T., Leruste, A., Dhingra, S., Martinez-Velez, N., Xu, P., Huang, J., Delaidelli, A., Desai, M. H., Good, Z., Polak, R., May, A., Labanieh, L., Bjelajac, J., Murty, T., Ehlinger, Z., Mount, C. W., Chen, Y., Heitzeneder, S., Marjon, K. D., Banuelos, A., Khan, O., Wasserman, S. L., Spiegel, J. Y., Fernandez-Pol, S., Kuo, C. J., Sorensen, P. H., Monje, M., Majzner, R. G., Weissman, I. L., Sahaf, B., Sotillo, E., Cochran, J. R., Mackall, C. L. 2024

  Abstract

  Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

  View details for DOI 10.1038/s41586-024-07443-8

  View details for PubMedID 38750365

  View details for PubMedCentralID 4182950

• Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells. Developmental cell Fowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, D. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M. 2024

  Abstract

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

  View details for DOI 10.1016/j.devcel.2024.03.003

  View details for PubMedID 38569552

• CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance. Proceedings of the National Academy of Sciences of the United States of America Banuelos, A., Zhang, A., Berouti, H., Baez, M., Yılmaz, L., Georgeos, N., Marjon, K. D., Miyanishi, M., Weissman, I. L. 2024; 121 (5): e2318534121

  Abstract

  The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.

  View details for DOI 10.1073/pnas.2318534121

  View details for PubMedID 38261615

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature Kamber, R. A., Nishiga, Y., Morton, B., Banuelos, A. M., Barkal, A. A., Vences-Catalan, F., Gu, M., Fernandez, D., Seoane, J. A., Yao, D., Liu, K., Lin, S., Spees, K., Curtis, C., Jerby-Arnon, L., Weissman, I. L., Sage, J., Bassik, M. C. 2021

  Abstract

  Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

  View details for DOI 10.1038/s41586-021-03879-4

  View details for PubMedID 34497417

• Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance. Proceedings of the National Academy of Sciences of the United States of America Upton, R., Banuelos, A., Feng, D., Biswas, T., Kao, K., McKenna, K., Willingham, S., Ho, P. Y., Rosental, B., Tal, M. C., Raveh, T., Volkmer, J., Pegram, M. D., Weissman, I. L. 2021; 118 (29)

  Abstract

  Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-alpha (SIRPalpha) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

  View details for DOI 10.1073/pnas.2026849118

  View details for PubMedID 34257155

• Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome. Proceedings of the National Academy of Sciences of the United States of America Li, J., Brickler, T., Banuelos, A., Marjon, K., Shcherbina, A., Banerjee, S., Bian, J., Narayanan, C., Weissman, I. L., Chetty, S. 2021; 118 (15)

  Abstract

  Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a "don't eat me" signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic "eat me" signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.

  View details for DOI 10.1073/pnas.2005483118

  View details for PubMedID 33833053

• Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, J., Brickler, T., Banuelos, A., Marjon, K., Shcherbina, A., Banerjee, S., Bian, J., Narayanan, C., Weissman, I. L., Chetty, S. 2021; 118 (15)

  View details for DOI 10.1073/pnas.2005483118|1of11

  View details for Web of Science ID 000641176100027

• Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+breast cancer (BC) xenografts growth in vivo Upton, R., Feng, D., Banuelos, A. M., Biswas, T., Willingham, S., Kao, K. S., McKenna, K., Rosenthal, B., Tal, M. C., Volkmer, J., Pegram, M. D., Weissman, I. L. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000618737700575

• Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells. eLife Zaro, B. W., Noh, J. J., Mascetti, V. L., Demeter, J., George, B., Zukowska, M., Gulati, G. S., Sinha, R., Flynn, R. A., Banuelos, A., Zhang, A., Wilkinson, A. C., Jackson, P., Weissman, I. L. 2020; 9

  Abstract

  The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

  View details for DOI 10.7554/eLife.62210

  View details for PubMedID 33236985

• GD2 is a macrophage checkpoint molecule and combined GD2/CD47 blockade results in synergistic effects and tumor clearance in xenograft models of neuroblastoma and osteosarcoma Theruvath, J., Smith, B., Linde, M. H., Sotillo, E., Heitzeneder, S., Marjon, K., Tousley, A., Lattin, J., Banuelos, A., Dhingra, S., Murty, S., Mackall, C. L., Majzner, R. G. AMER ASSOC CANCER RESEARCH. 2020: 35

  View details for Web of Science ID 000551367400035

• Overexpression of CD47 is associated with brain overgrowth in 16p11.2 deletion syndrome bioRxiv Li, J., Brickler, T., Banuelos, A., Marjon, K., Bian, J., Narayanan, C., Weissman, I. L., Chetty, S. 2019

  View details for DOI 10.1101/808022

• Mass spectrometry analysis of mouse hematopoietic stem cells and their progenitors reveals differential expression within and between proteome and transcriptome throughout adult and aged hematopoiesis bioRxiv Zaro, B. W., Noh, J. J., Mascetti, V. L., Demeter, J., George, B. M., Zukowska, M., Gulati, G. S., Sinha, R., Morganti, R. M., Banuelos, A. M., Zhang, A., Jackson, P. K., Weissman, I. L. 2019

  View details for DOI 10.1101/836692

• Programmed cell removal by calreticulin in tissue homeostasis and cancer NATURE COMMUNICATIONS Feng, M., Marjon, K. D., Zhu, F., Weissman-Tsukamoto, R., Levett, A., Sullivan, K., Kao, K. S., Markovic, M., Bump, P. A., Jackson, H. M., Choi, T. S., Chen, J., Banuelos, A. M., Liu, J., Gip, P., Cheng, L., Wang, D., Weissman, I. L. 2018; 9

  View details for DOI 10.1038/s41467-018-05211-7

  View details for Web of Science ID 000441306000001

• Programmed cell removal by calreticulin in tissue homeostasis and cancer. Nature communications Feng, M. n., Marjon, K. D., Zhu, F. n., Weissman-Tsukamoto, R. n., Levett, A. n., Sullivan, K. n., Kao, K. S., Markovic, M. n., Bump, P. A., Jackson, H. M., Choi, T. S., Chen, J. n., Banuelos, A. M., Liu, J. n., Gip, P. n., Cheng, L. n., Wang, D. n., Weissman, I. L. 2018; 9 (1): 3194

  Abstract

  Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

  View details for PubMedID 30097573