Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression

Adequate hematopoiesis regeneration under stress determines the disease outcome and is governed by bone marrow (BM) hematopoietic stem cell (HSC) niche, which controls HSC self-renewal, differentiation, and migration.^1-3 The niche is composed of cellular components, including mesenchymal stem cells (MSCs), mesenchymal progenitor cells (MPCs), mature lineages like osteoblasts and adipocytes,^4-6 endothelial cells (ECs), and the factors produced by them. These cells can be identified based on their marker expression by fluorescence-activated cell sorting (FACS)^5,7-9 and confocal imaging.¹⁰ Extracellular matrix (ECM) proteins represent an important family of molecular niche components, forming a complex structural network but also transducing signals via cellular receptors. Nevertheless, so far, most studies have been focusing on the role of the cellular niches.^5,11,12 Little is known about the role of ECM molecules in controlling hematopoiesis and leukemia.

Laminins are a large family of heterotrimeric ECM glycoproteins composed of α, β, and γ chains. The laminin α chain contains major binding sites for their receptors,^13,14 and thus, is crucial for the functional activities of the laminins.¹⁵ Laminin α4 (LAMA4), the active chain for several laminin isoforms (laminin-411, -421, and -423), is expressed in BM and plays a role in HSC proliferation and migration.^16-18 Deletion of Lama4 in mice led to disruption of angiogenesis perinatally¹⁹ but had a minor impact on hematopoiesis under steady-state.¹⁶ However, the impact of Lama4 on hematopoiesis regeneration and leukemia progression remains largely unclear.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a poor survival rate.²⁰ Progressive loss of normal hematopoiesis is directly associated with morbidity and mortality in leukemias.²¹ The hematopoietic insult induced by radiotherapy or chemotherapy further increases the risk for death due to pancytopenia. Thus, quick and efficient hematopoiesis recovery is essential for successful treatment outcomes. It has been shown that AML cells can alter BM niche and render the niche more supportive for AML cells but hostile for normal hematopoiesis.^5,22 Furthermore, emerging evidence suggests that BM stromal cells protect leukemic cells from chemotherapy by providing metabolic support^23-29 and maintaining them in quiescence.³⁰ However, the molecular mechanisms remain to be elucidated.

We have shown that Lama4 is upregulated in mouse BM MSCs and MPCs but not ECs during AML progression.⁵ However, the functional impact of the LAMA4 expression alteration on AML has been unexplored. Here, we have used Lama4^−/− mice and an MLL-AF9 AML model and demonstrated that Lama4 deficiency caused impaired hematopoiesis recovery, accelerated AML progression, and relapse following treatment, demonstrating a critical role of Lama4 in maintaining normal hematopoiesis while suppressing AML. Importantly, LAMA4 inhibition or knockdown in human BM MSCs had a similar impact on human AML cell proliferation and protection. Mechanistically, in addition to altered cellular niche composition in Lama4^−/− BM, upregulated genes for inflammatory response were detected in Lama4^−/− MSCs and MPCs. Upon AML exposure, Lama4^−/− MSCs seemed to be more susceptible to remodeling by AML cells. They provided a stronger antioxidant defense through increased mitochondrial transfer to the AML cells, thereby contributing to increased AML proliferation and chemoprotection.

Wild type (Lama4^+/+) and Lama4^−/− CD45.2 C57BL/6J mice¹⁹ aged 7 to 14 weeks were used for this study. Transplantation-induced MLL-AF9 AML mouse model was generated as described.⁵ Animal procedures were performed with approval (2018-1869) from the local ethics committee at Karolinska Institute (Stockholm, Sweden). See more details in the supplemental Data, available on the Blood Web site.

BM MSCs or MPCs were sorted, and RNA was extracted using RNeasy MicroKit (Qiagen) according to the manufacturer’s recommendations. cDNA was prepared using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). The cDNA quality was examined on the Agilent TapeStation system using a High Sensitivity D5000 ScreenTape (Agilent). One ng cDNA was used for library preparation using Nextera XT DNA Library Preparation Kit (FC-131-1024 & FC-131-1096, Illumina). The yield and quality of the amplified libraries were analyzed using Qubit (Thermo Fisher Scientific) and the Agilent TapeStation System. The indexed cDNA libraries were normalized, combined, and sequenced on the Illumina Nextseq 550 or 2000 for a 75-cycle v2 or 130-cycle P2 sequencing run generating 75 bp single-end or pair-end reads. See supplemental Data for data analysis.

The cultured or freshly isolated cells were washed with phosphate-buffered saline (PBS) and incubated with 2 µM of H₂-DCFDA (C6827, ThermoFisher) or 4 nM tetramethylrhodamine methyl ester perchlorate (TMRM) (9105, Immunochemistry Technologies) in DMEM at 37°C for 40 minutes to detect ROS and MMP level, respectively. The cells were then rinsed twice with PBS and resuspended with 150 µL of propidium iodide (1:1000) in 5% fetal bovine serum/PBS. ROS and MMP levels were measured by FACS.

MSCs were stained with MitoTracker™ Green FM (M7514, Thermo Fisher) at 37°C for 60 minutes, according to the manufacturer’s instructions. The cells were washed twice with PBS, then incubated for 3 to 4 hours to remove the unbound probe before a final wash. Subsequently, 15 000 to 30 000 MLL-AF9 AML cells in Myelocult (M5300, StemCell Technologies) were plated and cocultured with prelabeled MSCs for 24 hours. Mitochondrial transfer was quantified in AML cells (CD45.1⁺) by FACS and visualized by confocal microscope.

Unpaired Student t test or Mann-Whitney U test was used to determine the differences based on the data distribution. The Kaplan-Meier survival curves of the mice were generated by Log-rank (Mantel-Cox test) in Prism 8.0. All reported P values were obtained using the Prism 8.0 software, and P < .05 was considered statistically significant.

See additional methods including confocal imaging, colony assays, and MSC isolation in the supplemental Data.

We determined LAMA4 distribution in BM vascular structure by confocal imaging. We observed colocalization of LAMA4 with SCA1 and endomucin, markers for arteriolar and sinusoidal vessels, respectively, indicating that LAMA4 is expressed in arteriolar and sinusoidal vessels. Scattered expression of LAMA4 was detected in intersinusoidal spaces of BM (Figure 1A-B). As expected, no LAMA4 was detected in Lama4^−/− BM (Figure 1C), showing the specificity of the LAMA4 antibody staining. To examine Lama4 expression in specific BM cellular niches, we performed quantitative PCR (Q-PCR) of FACS-sorted MSCs (CD45⁻LIN⁻CD31⁻CD44⁻CD51⁺SCA1⁺), MPCs (CD45⁻LIN⁻CD31⁻CD44⁻CD51⁺SCA1⁻), and ECs (CD45⁻LIN⁻CD31⁺) from mouse BM. Notably, Lama4 mRNA was expressed in Lama4^+/+ BM MSCs and MPCs, even at a higher level than that in ECs (Figure 1D-E). Low or undetectable expression was found in mature stromal cells (CD44⁻SCA1⁻CD51⁻ and CD44⁺) and CD45⁺/TER119⁺ hematopoietic cells (Figure 1E).

The effect of Lama4 deletion on hematopoietic lineage production remains to be examined. Under steady-state, we detected lower platelets (PLTs) and reduced frequency of granulocytes (CD11B⁺GR1⁺) but no significant changes in white blood cells and red blood cells (RBCs) in peripheral blood (PB) of Lama4^−/− mice (supplemental Figure 1). There was no difference in BM cellularity and frequency of hematopoietic stem and progenitor cells (HSPCs) in Lama4^+/+ and Lama4^−/− mouse BM (supplemental Figure 2A-D).

To study the role of Lama4 in hematopoiesis under stress, we analyzed hematopoiesis recovery after irradiation (7Gy) in Lama4^−/− and Lama4^+/+ mice (supplemental Figure 1A). The Lama4^−/− mice displayed a slower recovery of RBCs and PLTs in PB (supplemental Figure 1B). The increased mean corpuscular volume of Lama4^−/− RBCs suggested defective maturation. FACS analysis showed reduced numbers of granulocytes (CD11B⁺GR1⁺) but increased T cells in Lama4^−/− PB (supplemental Figure 1C). In addition, we observed slower regeneration of granulocyte-macrophage progenitors and megakaryocyte progenitors (MkPs), but higher frequencies of megakaryocyte-erythrocyte progenitors and erythrocyte precursor (CFU-E) in Lama4^−/− BM and spleen at 4 to 6 weeks postirradiation (supplemental Figure 1D-E). The phenotypically defined HSCs remained unchanged (supplemental Figure 1D). The changes in CFU-E and CFU-GM were confirmed by CFU-C assays and further supported by the increased spleen cellularity (supplemental Figures 1E and 3A-C). This finding, together with the reduced production of mature RBCs and PLTs, points to a possible differentiation block of erythropoiesis and megakaryopoiesis between the intermediate progenitors and the mature lineages. Taken together, these data suggest that Lama4 is important for efficient hematopoiesis regeneration following irradiation-induced stress.

We have recently found that Lama4 is upregulated in BM MSCs and MPCs in AML mice.⁵ Data mining in Prognoscan³¹ suggests that lower expression of LAMA4 in BM mononuclear cells (MNCs) in the patients with AML correlates with poor survival rate (supplemental Figure 4). However, the functional impact on AML progression remains unknown. To investigate this, we employed a transplantation-induced MLL-AF9 AML mouse model without prior irradiation (Figure 2A). Interestingly, Lama4^−/−-recipient mice developed AML faster than Lama4^+/+ mice, reflected in the earlier onset of symptomatic AML, reduced survival rate, and increased AML burden in BM and PB (Figure 2B-C,E; supplemental Figure 5). The AML-initiating stem cells (LSCs, KIT⁺)³² were significantly increased in Lama4^−/− mouse BM and PB relative to that in Lama4^+/+ mice (Figure 2D-E; supplemental Figure 5C). Concurrently, Lama4^−/− mice showed more pronounced splenomegaly (Figure 2F), and their normal hematopoiesis seemed to be further decreased although not statistically significant (Figure 2G; supplemental Figure 5D-F). Lama4^−/− mice had increased egress of leukemic cells and normal HSPCs in PB (Figure 2H), as well as more severe reduction in PLTs (Figure 2I) than Lama4^+/+ mice. The PLT width (PDW) was correspondingly increased, indicating further impaired PLT maturation in the Lama4^−/− mice during AML progression (Figure 2I). Notably, we did not observe any homing advantage of the AML cells into Lama4^−/− recipient BM and spleen (Figure 2J-K; supplemental Figure 5H), pointing to enhanced proliferation rather than homing of AML cells that caused AML acceleration in Lama4^−/− mice.

To test the impact of Lama4^−/− environment on AML relapse, we transplanted normal BM MNCs into lethally irradiated Lama4^−/− and Lama4^+/+ mice after they developed AML to mimic clinical therapeutic transplantation (Figure 2L). We observed a faster relapse of AML in the Lama4^−/− mice, as shown by earlier recurrence of symptoms and reduced survival (Figure 2M-N). Notably, reconstitution from normal donor BM in lethally irradiated Lama4^−/− mice was comparable to that in Lama4^+/+ recipients shortly posttransplantation (supplemental Figure 6A), excluding the possibility that the faster relapse of AML in Lama4^−/− mice was due to impaired donor cell homing and regeneration in Lama4^−/− mice. These data suggest Lama4 expression is required for maintaining normal hematopoiesis while restricting AML proliferation.

BM vasculature plays an important role in maintaining normal hematopoiesis by impacting hematopoietic cell proliferation, migration, and differentiation.³³ Lama4 deletion resulted in impaired microvessel maturation perinatally.¹⁹ We examined BM vascular structure to explore whether it is related to the hematopoiesis regeneration defects and AML progression in Lama4^−/− mice. We observed reduced vessel density in Lama4^−/− mice at steady-state and 6 weeks after irradiation (Figure 3A; supplemental Figure 7A-B). Concurrently, CD31 fluorescence intensity in Lama4^−/− BM was reduced (supplemental Figure 7C). No difference in vessel leakiness was detected using Evans blue (data not shown).

Mk maturation is controlled by their adhesive interactions with the vascular niche.^34-36 We analyzed the colocalization of Mk with vasculature in Lama4^−/− BM. While there was no difference in the total number of Mk in Lama4^−/− mouse BM, the Mks adjacent to sinusoidal vessels were reduced in Lama4^−/− mouse BM by about 20% 6 weeks after irradiation (Figure 3A,C). Such a reduction was not observed at steady-state (Figure 3C). To examine whether this defect could be rescued by exogenous LAMA4 proteins, we cultured Lama4^−/− marrow or LIN⁻SCA1⁻CD41⁺CD150⁺ MkPs with recombinant LAMA4 (Figure 3D). We observed increased Mk maturation after culture with LAMA4, reflected in a higher proportion of CD41⁺ cells with larger size and higher granularity (Figure 3E-J). Altogether, our data suggest LAMA4 plays a role in Mk maturation.

BM MSCs and MPCs are critical for maintaining normal hematopoiesis and regulating myeloid malignancies.¹ Therefore, we next investigated the impact of Lama4 deletion on BM mesenchymal cell niches. FACS analysis showed reduced frequency of MPCs and ECs in Lama4^−/− mouse BM at steady-state (Figure 4A-B). However, the frequency of Lama4^−/− MSCs and mature stromal cells remained unchanged under steady state (Figure 4A-B; supplemental Figure 7E) and 6 weeks postsublethal irradiation (supplemental Figure 7F), which was supported by similar CFU-Fs in Lama4^+/+ and Lama4^−/− mouse BM (Figure 4C). To explore the molecular mechanisms contributing to the impaired hematopoiesis regeneration, we determined erythropoietin (EPO) level in blood plasma and found reduced EPO in Lama4^−/− mice 6 weeks postirradiation (supplemental Figure 7G). This might explain the impaired erythropoiesis in Lama4^−/− mice postirradiation. Additionally, Q-PCR showed no dramatic molecular alterations at steady-state in several genes known to be important for hematopoiesis in Lama4^−/− BM MSCs, MPCs, and ECs (Figure 4D). However, Angpt1 and Cxcl10 were downregulated, but Tgfb1 was upregulated in Lama4^−/− MPCs at 6 weeks postirradiation (Figure 4E). Thus, we decided to perform global gene expression profiling by RNA sequencing on these stressed BM MPCs.

RNA sequencing revealed distinct transcriptional profiles of the Lama4^+/+ and Lama4^−/− MPCs sorted from mice at 6 weeks postsublethal irradiation (supplemental Figure 8A). The Lama4 depletion led to an overall dysregulation of mRNA expression in MPC, with 381 downregulated genes and 59 upregulated genes in Lama4^−/− BM MPCs, compared with Lama4^+/+ MPCs (Figure 4F). As expected, Lama4 was the most downregulated gene in the Lama4^−/− MPCs (Figure 4F). Interestingly, several genes encoding ECM proteins that, together with laminins, are assembled to form the structural matrix network in BM^37,38 were downregulated. These include Fn1, Fbn1, Nid1, and thrombospondin-1 (Thbs1) (Figure 4F; supplemental Figure 8B-C). Meanwhile, genes associated with inflammatory response were upregulated in Lama4^−/− MPCs (Figure 4F-H; supplemental Figure 8D). Strikingly, while the genes related to oxidative phosphorylation were enriched in the Lama4^−/− MPCs, pointing to a possible enhanced ROS production, the genes related to ROS metabolic pathway and antioxidant activity were also upregulated in these cells (Figure 4G; supplemental Figure 8E-F). Furthermore, several proapoptotic genes were significantly upregulated (supplemental Figure 8G), although the functional consequence remained to be tested. In addition, we observed downregulation of CD49f/integrin α6 (Itga6), a main receptor for LAMA4 in Lama4^−/− MSCs and MPCs (supplemental Figures 8C and 9; Figure 4F). CD146, a newly identified LAMA4 receptor,¹³ was also reduced in steady-state Lama4^−/− ECs and stressed Lama4^−/− MSCs (supplemental Figure 9).

These data suggest that Lama4 deficiency alters BM cellular niche composition and molecular profile, which may result in abnormal niche support for normal hematopoiesis and AML.

To explore the functional contribution of Lama4^−/− BM MSCs to AML proliferation and therapy response, we performed cobblestone area-forming cell (CAFC) assay using Lama4^−/− and Lama4^+/+ BM MSCs in coculture with MLL-AF9 AML cells (Figure 5A). The AML cells were cultured with the MSCs for 2 days prior to adding Ara-C to allow for AML cell adhesion and migration. Following an additional 5 to 7 days, the AML LSCs were assessed functionally by CAFC counts and phenotypically by FACS for the cells expressing KIT or CD36, both of which have been reported to be LSC markers.^29,32,39 We observed more CAFCs in the culture with Lama4^−/− MSCs than with Lama4^+/+ MSCs, although the total numbers of AML cells were comparable (Figure 5B-D), suggesting enhanced support of Lama4^−/− MSCs for the growth of AML LSCs. Post-Ara-C treatment, more CAFCs, and KIT⁺ AML LSCs, but not CD36⁺ AML cells, were maintained in the culture with Lama4^−/− MSC compared with that Lama4^+/+ MSCs (Figure 5C-F). ROS level has been shown to be an important factor determining the survival of AML cells after chemotherapy.^27,29,40 Therefore, we analyzed ROS levels in the MSCs and AML cells in the coculture and observed lower ROS levels in Lama4^−/− MSCs and the AML cells cocultured with them than those in the Lama4^+/+ MSC cocultures (Figure 5G). These data suggest that Lama4^−/− MSCs could promote AML LSC proliferation and chemoprotection.

To explore if stroma-derived LAMA4 had any impact on human AML cell growth and therapy response, we employed function inhibition strategies by using anti-LAMA4 neutralizing antibodies (Figure 5H) or LAMA4 shRNA knocking down (Figure 5J-K). Similar to the finding in the mouse model, LAMA4 neutralization or shRNA knockdown in human BM MSCs increased human AML cell (THP1) proliferation and chemoresistance to Ara-C (Figure 5I,L). These data indicate a suppressive role of LAMA4 in human AML cell proliferation and therapy response.

It has been shown that niche-remodeling by leukemia may contribute to forming a self-reinforcing niche that promotes leukemia progression but impairs normal hematopoiesis. We hypothesized that Lama4^−/− stroma could be more susceptible to the remodeling by AML cells. To test this, we exposed Lama4^+/+ and Lama4^−/− MSCs to the AML cells in vitro and determined their molecular response by RNA sequencing (Figure 6A). This revealed distinct gene expression profiles of Lama4^+/+ and Lama4^−/− MSCs before and after AML exposure (Figure 6B) with significantly more genes altered in Lama4^−/− MSCs than Lama4^+/+ MSCs (826 vs 229) post-AML exposure, indicating increased susceptibility of Lama4^−/− MSCs by AML (Figure 6C-D). Many genes, including genes associated with focal adhesion and inflammatory response, were uniquely altered in the Lama4^−/− MSCs post-AML exposure (Figure 6D-F). Among those, Lama5, Lama2, Lama1, and their receptors were upregulated in the Lama4^−/− MSCs, consistent with previous reports.^16,19 However, genes related to cytokine-cytokine receptor interactions were enriched in Lama4^−/− MSCs before AML exposure and did not alter further post-AML exposure. Glutathione metabolism plays an important role in preventing cellular damages induced by ROS, the level of which has been shown to be important for the survival of AML cells after chemotherapy.^27,29,40 Glutathione metabolism genes were enriched in Lama4^−/− MSCs (Figure 6H), which might be associated with the reduced ROS levels in Lama4^−/− MSCs (Figure 5G). Notably, some niche factors like Kitl, Il11, Cxcl12, and Spp1, known to be dysregulated in leukemia,^7,21,41-43 were more significantly altered in Lama4^−/− MSCs post-AML exposure than in Lama4^+/+ MSCs (Figure 6I). Among those, Kitl/Scf and Il11 have been shown to promote AML cell proliferation,³² while Angpt1 and Angpt2 were reported to be associated with therapy response and prognosis in leukemia.^44-47 In addition, Sipa1 loss in the BM niche could induce hematopoietic cell transformation.⁹ Lower Sipa1 expression was detected in Lama4^−/− MSCs. Along with these molecular alterations in the stroma, RNA sequencing of the AML cells cocultured with Lama4^−/− MSCs showed downregulation of cell proliferation inhibitors, including Egr1,^48,Neo1,⁴⁹ and arachidonic acid metabolism-associated genes (Figure 6J), which can be activated in response to oxidative stress.⁵⁰ These changes might have caused the enhanced AML proliferation in the culture with Lama4^−/− MSCs. Together, these data provide molecular evidence for the enhanced AML supportive and protective functions of Lama4^−/− MSCs.

To further determine the chemoprotective role of Lama4^−/− niche in vivo and the potential mechanism, we tested the therapy response of AML cells to Ara-C by injecting the MLL-AF9⁺ AML cells expressing fluorescence protein DsRed into sublethally irradiated Lama4^−/− mice. Lama4^−/−-recipient mice showed faster AML progression (Figure 7A; supplemental Figure 10). Following a 5-day Ara-C injection, all mice developed leukopenia (supplemental Figure 10B) and ameliorated splenomegaly, showing the efficacy of the treatment. However, the spleens in Lama4^−/− mice remained bigger than in Lama4^+/+ mice 1 day post-Ara-C treatment (Figure 7B). The residual AML cells and the enrichment of CD36⁺ cells appeared to be higher in Lama4^−/− mouse BM post-Ara-C treatment (Figure 7C). Similar to the findings in vitro, the ROS levels in the AML cells from Lama4^−/− recipient PB was reduced compared with that in Lama4^+/+ mice during AML development (Figure 7D). In contrast to the dramatic elevation of ROS in the AML cells from Lama4^+/+ recipient BM after Ara-C treatment, ROS levels remained unchanged in the AML cells from Lama4^−/− recipients (Figure 7E). These data suggest stronger antioxidant activities of Lama4^−/− niche for AML cells.

Mitochondrial transfer from BM MSCs to AML cells has been recognized as a mechanism of niche support of AML cells with antioxidant defense, and this transfer increases upon chemotherapy.^23-25,27 To examine whether mitochondrial transfer mediated the antioxidative stress effect of Lama4^−/− MSCs on AML cells, we cocultured MLL-AF9 AML cells with MSCs prelabeled with MitoTracker and analyzed MSC-derived mitochondria in AML 24 hours after coculture (Figure 7F). First, the Lama4^−/− MSCs prior to the coculture displayed increased mitochondrial mass and MMP, and correspondingly, lower ROS levels (Figure 7G-H). The mitochondrial mass in the Lama4^−/− MSCs remained higher after the coculture with AML cells compared with that in the Lama4^+/+ MSCs (Figure 7I), suggesting increased ROS-scavenging capacity in Lama4^−/− MSCs. Interestingly, mitochondrial transfer from Lama4^−/− MSCs to the AML cell was increased compared with that from Lama4^+/+ MSCs during Ara-C treatment in the coculture (Figure 7J-K). However, this was not observed in the cultures without Ara-C (Figure 7L). The mitochondrial transfer was mostly mediated by direct cell interactions since it diminished by 90% without cell contact (supplemental Figure 11). These data together indicate that Lama4^−/− MSCs provide stronger metabolic support for AML cells by donating more mitochondrial antioxidant tools.

Here we have, for the first time, demonstrated that Lama4-deficient environment accelerates AML progression and relapse. In addition, Lama4 deficiency in BM MSCs promotes proliferation and drug resistance of the AML LSCs in vitro. Importantly, the finding holds true also in human AML cells cocultured with human MSCs upon LAMA4 antibody inhibition and knockdown. Mechanistically, the protective role of Lama4^−/− niche seems to be associated with enhanced ROS-scavenging activity of Lama4^−/− cellular niches and mitochondrial transfer from the MSCs to AML cells, which could contribute by detoxifying chemotherapy-induced ROS overproduction in the AML cells. Our study provides the first evidence for the critical role of Lama4 for hematopoiesis regeneration under stress and AML progression.

Although LAMA4 was shown to be mainly expressed in BM vasculature in mice,^16,51,52 its expression in BM mesenchymal cell niches remained unknown. We here describe Lama4 expression in mouse BM MSCs and MPCs, opening a possibility that LAMA4 may mediate crosstalk between mesenchymal cell niches and HSPCs that are known to express LAMA4 receptor integrin α6/CD49F.^53,54

The impact of LAMA4 on BM niche maintenance is demonstrated by the phenotypic and molecular alterations in BM ECs and MPCs in Lama4^−/− mice. The molecular niche alterations in Lama4^−/− mice, particularly the enhanced inflammatory response that is reported to negatively regulate normal hematopoiesis,^11,55,56 might result in the impaired hematopoiesis regeneration observed here following irradiation and accelerate AML development. BM sinusoidal ECs provide not only a cellular platform whereby HSPCs migrate and reconstitute but also a conduit for the release of mature hematopoietic cells to the circulation.^57-60 The reduced vessel-associated megakaryocytes in Lama4^−/− BM after the irradiation could affect Mk transendothelial protrusion and maturation. Our finding that LAMA4 proteins increased Mk maturation in vitro support this notion.

We here, for the first time, demonstrated the critical role of Lama4 in AML progression and drug response. Deletion of Lama4 accelerated AML development and relapse. Importantly, inhibition/knockdown of LAMA4 in human BM MSCs also promoted human AML cell proliferation and chemoprotection, warranting future studies on the potential therapeutic effects of LAMA4 proteins in patient AML cells.

Drug resistance is the main challenge for the successful treatment of AML. Recently, increasing evidence has indicated the contribution of BM stromal cells to chemoresistance of leukemic cells by regulating metabolic activities of the leukemic cells via directly donating mitochondrial transfer to leukemic blasts or indirectly altering the balance between ROS production and detoxification.^23,24,28 An important finding in our study is that Lama4^−/− MSCs confer AML LSC chemoresistance. Mechanistically, Lama4^−/− MSCs displayed enhanced glutathione redox activities and a substantial mitochondrial transfer into the AML cells. This is accompanied by the reduced ROS level in the AML cells from Lama4^−/− niche, presenting a possible mechanism that Lama4^−/− niches balance ROS levels in AML cells, thereby attenuating the chemotherapeutic effects from Ara-C on the AML cells. This notion is supported by the increased mitochondrial mass and the enriched genes related to ROS-scavenging activity in Lama4^−/− MSCs.

In summary, our study presents the first evidence for the impact of Lama4 on hematopoiesis regeneration and AML progression. Lama4^−/− mesenchymal niches promote AML progression and increase drug resistance, possibly by providing antioxidant support via increased mitochondrial transfer. Therefore, strategies to reactivate LAMA4 receptor-signaling pathways may be a promising therapeutic approach to suppress AML and sensitize AML LSCs to chemotherapy, thereby improving therapeutic efficacy for AML.

This study was supported by the Swedish Research Council (2019-01361), Swedish Childhood Society (PR2015-0142, FoAss13/015, and PR2017/0154, PR2020-0166), Swedish Cancer Society (CAN2017/774, 19 0092 SIA, and 20 1222 PjF), Åke Olsson Foundation, Radiumhemmets Forskningsfonder and Karolinska Institute, Wallenberg Institute for Regenerative Medicine, Karolinska Institute doctoral education (KID) funding (2-1293/2014), Stiftelsen Clas Groschinskys Minnesfond (ref:M16 50) to H.Q. FACS analysis and cell sorting were performed at MedH Flow Cytometry core facility (Karolinska Institute), supported by KI/SLL. The confocal images were obtained at the LCI facility/Nikon Center of Excellence, Karolinska Institutet, supported by grants from the Knut and Alice Wallenberg Foundation, Swedish Research Council, KI infrastructure, Centre for Innovative Medicine, and Jonasson Center at the Royal Institute of Technology. All major computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under Project sens2018540. The authors also thank the core facility at NEO, BEA, Bioinformatics, and Expression Analysis, which is supported by the board of research at the Karolinska Institute and the research committee at the Karolinska hospital.

Contribution: H.Q., M.K., and H.C. have participated in conception and design, performing experiments, collection and assembly of data, data analysis and interpretation, and manuscript writing; L.S., P.X., and A.-S.J. performed experiments, collected data, and assisted with data analysis; K.T. provided Lama4^−/− mice; T.S. provided antibodies against laminin chains; J.Z.-P. assisted shRNA knockdown experiments; J.U. provided human samples and scientific input; M.E. and J.W. provided scientific input/advice and manuscript-reviewing; and all authors have proved the final version of the manuscript.

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

The current affiliation for M.K. is Cell Sheet Tissue Engineering Center, Department of Pharmaceutics and Pharmaceutical Chemistry, Health Sciences, University of Utah, Salt Lake City, UT.

Correspondence: Hong Qian, Center for Hematology and Regenerative Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital, SE-141 86 Stockholm, Sweden; e-mail: hong.qian@ki.se.