CD99 tunes translation to sustain stem cells Free

In this issue of Blood, Ji et al¹ demonstrate that CD99 plays an important role in maintaining the self-renewal and function of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) by maintaining low rates of protein synthesis. These insights reveal a mechanism that promotes the HSC regenerative capacity by reducing proteotoxic stress and highlight a potential vulnerability in LSCs that may be targeted for therapeutic benefit.

Protein synthesis in HSCs varies significantly across different developmental and disease conditions. In adult HSCs, protein synthesis rates are relatively low and tightly regulated to preserve self-renewal and long-term regenerative potential.² Disruptions to this balance by genetic perturbations or in disease states impair HSC function.³ Excessive protein synthesis overwhelms the cell’s protein folding machinery, leading to the accumulation of unfolded or misfolded proteins, which activate the unfolded protein response (UPR) and the endoplasmic reticulum–associated degradation (ERAD) pathways, which are aimed at restoring cellular homeostasis. The depletion of chaperones, UPR sensors, or ERAD components impairs HSC self-renewal and regenerative capacity. In addition, heat shock protein (HSF1), a key regulator of the cellular response to stress, promotes proteostasis and increases HSC fitness in response to ex vivo culture stress and aging.⁴ 

The ribosome biogenesis and protein synthesis rates are highly context-dependent. Elevated protein synthesis rates, when compared with healthy hematopoietic stem and progenitor cells (HSPCs), have been observed in acute myeloid leukemia (AML) cell lines and primary AML LSCs from mouse models and patient samples, and inhibiting protein synthesis can effectively suppress leukemia cell growth in some contexts.^5-7 Conversely, AML cells that harbor loss-of-function mutations in RUNX1 showed impaired ribosome biogenesis and likely reduced protein synthesis, thereby making them more sensitive to protein translation inhibitors.⁸ These findings highlight the intricate and complex nature of protein synthesis in normal and diseased hematopoietic cells.

Previous data showed that CD99 is highly expressed in stem cells from myelodysplastic syndrome and functional LSCs in AML,^9,10 and blocking CD99 function using an anti-CD99 monoclonal antibody significantly blunted AML progression, indicating its potential role in AML development.⁹ Notably, they also observed a marked reduction in translation-associated gene signatures in CD99^high LSCs, implying a potential role of CD99 in regulating protein synthesis.

To explore this further, Ji et al used a CD99-deficient mouse model to examine its effects on global protein translation and its role in regulating HSC and LSC function. Although dispensable for steady state hematopoiesis, CD99 deficiency significantly impaired HSC regeneration after extensive proliferative stress from secondary transplantation. To explore the underlying mechanisms, they performed RNA sequencing and identified that the mTORC1/ribosome protein genes and the UPR pathway genes were among the most upregulated signatures in CD99^–/– HSCs. Indeed, CD99 loss led to an aberrant increase in protein synthesis in proliferating CD99^–/– HSCs. The authors then applied 2 chemical inhibitors, namely rapamycin that target mTORC1 or CX-5461 that target RNA polymerase I, thereby inhibiting ribosome biogenesis, and reduced protein synthesis genetically by crossing CD99^–/– mice with RPL24^Bst/+ mice to assess if restoring the protein synthesis balance could rescue HSC function. All 3 approaches restored the CD99^–/– HSC regenerative capacity in transplantation assays. Furthermore, CD99 deficiency increased the levels of ubiquitinated proteins and triggered the integrated stress response upon proliferation-induced proteotoxic stress. The nuclear accumulation of HSF1 in HSCs under bortezomib-induced proteotoxic stress is indicative of UPR activation. Treatment with 17-AAG (17-N-allylamino-17-demethoxygeldanamycin), a HSP90 inhibitor and HSF1 activator, restored the CD99^–/– HSC function to wild-type levels by reducing the rates of protein synthesis.

Having uncovered the novel function of CD99 in constraining the rate of protein synthesis to maintain proteostasis to support HSC regeneration, the authors next explored LSCs. They found that AMLs that harbored AML-ETO mutations expressed high levels of CD99. In an AML1-ETO–driven AML mouse model, depletion of CD99 increased the rate of protein synthesis and impaired the leukemogenic potential of LSCs in secondary transplantation experiments. Pharmacologic treatment with rapamycin reduced the rate of protein synthesis, promoted proteostasis, and rescued LSC function in CD99^–/– mice while it exerted opposite effects on wild-type LSCs. This genotype-dependent differential responses of LSCs to rapamycin implicate the complexity of the regulation of protein translation, stress response, and LSC self-renewal. Taken together, these results suggest that both HSCs and LSCs share mechanisms centered on tightly regulated protein translation and proteostasis to maintain their identity and function.

Understanding the regulatory networks that govern proteostasis under various physiological and pathologic conditions is crucial for developing antiaging strategies and therapies for hematologic disorders. The study uncovered a novel role for the surface protein CD99 in regulating HSC function by maintaining the optimal protein synthesis levels and preventing proteotoxic stress. Several compelling questions arise from these findings and warrant further investigation. For instance, what regulates CD99 gene expression, protein levels, and surface expression? What are the physiologic ligands of CD99, especially in HSCs and LSCs? Does CD99 regulate proteostasis in AMLs without the AML-ETO translocation? Although it is known that CD99 negatively regulates SFK/Src signaling, how CD99 regulates mTOR and protein synthesis remains to be determined. To what extent does altered ribosome biogenesis contribute to CD99^–/– HSCs dysfunction? Because HSP90 has various client proteins in a cell, other mechanisms beyond HSF1 are likely involved in the complex responses observed upon 17-AAG treatment. CX-5461 is known to induce DNA damage, in addition to its purported role in inhibiting ribosome biogenesis; thus, caution should be taken when interpreting results involving this inhibitor. Considering the rapid proliferation of bulk leukemia cells and the need to constrain the protein synthesis rates to maintain proteostasis in LSCs, what is the optimal level of protein synthesis in LSCs, and does it differ from normal HSCs? Understanding this will be key to developing strategies to target LSCs and leukemia blasts while sparing normal HSPCs. Furthermore, addressing these questions will deepen our understanding of how CD99 influences hematopoietic regulation across different contexts and will ultimately shed light on how proteostasis can be manipulated to promote normal cell function while abrogating diseases.

Conflict-of-interest disclosure: The authors declare no competing financial interests.