CD38 emerges as a key regulator of tumor growth in multiple myeloma

Background MM is a malignant plasma cell disease characterized by high expression of CD38
.

Although monoclonal antibodies targeting CD38 are highly effective, drug resistance can develop
.

Tumor CD38 expression levels decreased after anti-CD38 mAb treatment, but sustained low expression was rarely seen
.

This suggests that CD38 expression may favor tumor cell survival, and the direct effect of CD38 deletion on tumor kinetics is unclear
.

The research method utilizes CRISPR-Cas9 gene editing technology to generate CD38 knockout (KO) cell lines
.

Non-targeted (NT) or CD38-KO J558 cells were injected subcutaneously into immune-normal BALB/c mice and immune-deficient NSG mice
.

The stromal adhesion of labeled NT and KO cells was compared with mouse bone marrow stromal cells (OP-9)
.

Intracellular nicotinamide adenine dinucleotide (NAD, coenzyme I) content was quantified using Promega Glo
.
Mitochondria were
isolated using a mitochondrial isolation kit (Thermo Scientific)
.

Oxygen consumption rate (OCR) was measured by a hippocampal metabolometer (Seahorse).
and extracellular acidification rate (ECAR) were quantified
.

Modular hypoxia chamber was used to assess cell response to hypoxia
.

Cell cycle was quantified by propidium iodide staining
.

As a result, we used CD38-expressing mouse plasma cells The tumor cell line J558 was used to study the role of CD38 in a mouse model
.

Strikingly, tumor volume was significantly reduced in BALB/c mice injected with CD38-KO cells compared to BALB/c mice injected with NT cells (113 mm3 (KO) vs 1293 mm3 (NT) at day 25, p <0.
001)
.

In contrast, cell proliferation and colony formation in KO and NT J558 cells were nearly identical in vitro, suggesting that the effects of CD38 deletion are highly context-dependent
.

Because tumor CD38 expression may negatively regulate immune responses, we next compared CD38-KO and NT cells injected into immunodeficient NSG mice
.

The tumor volume of mice injected with CD38 KOs cells was significantly reduced by about 2.
2-fold compared with those injected with NT cells (708 mm3 [KO] vs 1592 mm3 [NT], p=0.
07) (Fig.
1)
.

Figure 1 The growth of CD38 KO tumor cells was significantly reduced in vivo, and the role of CD38 in the immune microenvironment was indistinguishable in vitro
.

Considering that there are still some tumor growth barriers in immunodeficient mice, we next used J558 as well as human MM cell lines RPMI-8226 and NCI-H929 to study the effect of CD38 deletion on other aspects of cell proliferation
.

Daratumumab-induced CD38 internalization has been shown to reduce matrix adhesion of MM cells
.

Likewise, CD38 KO cells also showed reduced matrix adhesion (2.
5-fold for J558, p<0.
005; 2-fold for H929, p<0.
005)
.

Although stroma is a known promoter of cell survival and proliferation, we remain questionable whether the NAD metabolic activity of CD38 regulates tumor growth
.

CD38 overexpression reduces intracellular NAD levels and affects mitochondrial biosynthesis in vivo
.

At the same time, we found that the NAD level of KO J558 tumor cells was significantly higher than that of NT cells (2-fold change, p<0.
05) (Fig.
2)
.

Figure 2 Increased NAD+ and mitochondria in CD38 KO MM cells In addition, the mitochondrial protein levels in CD38 KO cells were significantly higher than those in NTs cells (5-fold for J558 and 2-fold for H929)
.

Metabolic activity of the CD38 KO cell line was also significantly increased, with a nearly 2-fold increase in basal OCR and ECAR as well as additional respiratory and glycolytic capacity (Fig.
3)
.

Figure 3 CD38 KO cells show elevated OXPHOS Given the differences in tumor growth ability in vivo and in vitro, it remains questionable whether changes in mitochondrial content and metabolic function can give CD38-expressing cells an advantage under hypoxic conditions.
is an important feature of the tumor microenvironment
.

Notably, under hypoxic (as opposed to very oxygen) conditions, CD38 KO MM cells exhibited significantly more cell cycle arrest, defined as G0/G1 arrest (p=0.
003 compared to H929; than RPMI, p=0.
004) (Fig.
4)
.

Figure 4.
CD38 KO cell proliferation is reduced only in hypoxia.
Conclusions Our study provides a new explanation for the role of CD38 in regulating the metabolism and proliferative potential of malignant plasma cells in hypoxic environment
.

In hypoxic conditions, CD38 deletion reduced tumor cell proliferation but enhanced stem cell properties
.

The phenotypic features of CD38 deletion can be explained by altered cellular metabolism (eg, higher OXPHOS)
.

Future research will aim to understand how inhibition of CD38 activity leads to metabolic alterations that could lead to therapeutic possibilities, leading to novel combination therapies of anti-CD38 mAbs or novel CD38 inhibitors
.

References: 1.
Ruxandra Maria Irimia, et al.
CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells.
2021 ASH POSTER 2652.
https://ash.
confex.
com/ash/2021/webprogram /Paper148693.
htmlEND